Method for producing recombinant antibody and antibody fragment thereof

ABSTRACT

A recombinant antibody or the antibody fragment thereof which specifically reacts with an extracellular domain of human CCR4; a DNA which encodes the recombinant antibody or the antibody fragment thereof; a method for producing the recombinant antibody or the antibody fragment thereof; a method for immunologically detecting CCR4, a method for immunologically detecting a cell which expressed CCR4 on the cell surface, a method for depleting a cell which expresses CCR4 on the cell surface, and a method for inhibiting production of Th2 cytokine, which comprise using the recombinant antibody according or antibody fragment thereof; a therapeutic or diagnostic agent for Th2-mediated immune diseases; and a therapeutic or diagnostic agent for a blood cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/684,651 (now U.S. Pat. No. 8,197,814); which is a Divisional of U.S.application Ser. No. 11/094,718, filed Mar. 31, 2005 (now U.S. Pat. No.7,666,418); which is a Divisional of U.S. application Ser. No.09/796,744, filed Mar. 2, 2001 (now U.S. Pat. No. 6,989,145); claimingpriority from JP 2000-59508, issued Mar. 3, 2000, and JP 2000-401563,issued Dec. 28, 2000; the entire disclosures of each of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a recombinant antibody and an antibodyfragment thereof which specifically react with the extracellular domainof human CC chemokine receptor 4 (hereinafter referred to as “CCR4”).Furthermore, the present invention relates to a recombinant antibodysuch as humanized antibody, human antibody and the like, and an antibodyfragment thereof which specifically react with CCR4, have cytotoxicactivity and activity of inhibiting production of cytokine by Th2 cells,and comprise a specific complementarity determining region (hereinafterreferred to as “CDR”). Moreover, the present invention relates to a DNAencoding the above mentioned antibody. Also, the present inventionrelates to a vector comprising the DNA, and a transformant transformedwith the vector. Still furthermore, the present invention relates to amethod for producing the above mentioned antibody using thetransformant, and a medicament such as a therapeutic agent, a diagnosticagent and the like, for Th2-mediated immune diseases such as allergicdiseases and the like, which comprises using the antibody. Stillmoreover, the present invention relates to a medicament such as atherapeutic agent, a diagnostic agent and the like, for cancers such asblood cancers, e.g., leukemia, which comprises using the antibody.

2. Brief Description of the Background Art

Various factors such as eosinophils, mast cells, IgE and the like,relate to allergic diseases such as bronchial asthma. Eosinophilsinfiltrate into an inflammatory part, release a cytotoxic basic proteinsuch as MBP (major basic protein) or the like, by degranulation tothereby induce injury of surrounding tissues. Mast cells releasehistamine by binding to an antigen immune complex with IgE produced fromB cells to thereby induce an immediate allergic reaction. They arecontrolled by biologically functional molecules such as cytokine,chemokine, and the like, which take part in signal transduction betweencells. Eosinophils are subjected to differentiation induction and lifespan prolongation by IL-5, and degranulation is further induced. IgE isproduced from B cells activated by IL-4 and becomes an immune complexwith the antigen to accelerate degranulation of mast cells. It has beenfound that IL-4, IL-13 and the like are also produced from mast cellsand contribute to the production of IgE from B cells, and the presenceof an allergy reinforcing loop has been confirmed (Am. J. Respir. Crit.Care Med., 152: 2059 (1995), Immunol. Today, 15: 19 (1994)). Thus, anelaborately cytokine-chemokine network is present between inflammatorycells and keeps complicated balances.

The cytokine and chemokine are produced by helper T cells which expressCD4 on the cell surface (hereinafter referred to as “CD4+Th cells”).Actually, it has been found that infiltration of helper T cells isremarkably found in the airway inflammation part of bronchial asthmapatients, wherein a considerably large number of the T cells areactivated and that the degree of seriousness and airway hypersensitivityof asthma correlates with the number of activated T cells, as well asthe activated T cells are also increased in the peripheral blood (Am.Rev. Respir. Dis., 145: S22 (1992)).

The helper T cells are classified into Th1 cells and Th2 cells,depending on the kind of cytokine to be produced thereby (Annu. Rev.Immunol., 7: 145 (1989)). Examples of the cytokine produced by Th2 cellsinclude IL-4, IL-5, IL-13, and the like.

It has been found that an antigen-specific T cell clone isolated from anatopic disease patient releases Th2 cytokine when stimulated in vitro(Proc. Natl. Acad. Sci., U.S.A., 88: 4538 (1991)), and Th2 cells arepresent in bronchoalveolar lavage fluid (hereinafter referred to as“BAL”) and airway mucosa of asthma patients (N. Engl. J. Med., 326: 298(1992), Eur. J. Immunol., 23: 1445 (1993)). IL-4 and IL-5 of Th2cytokines are increased when the expression of mRNA in BAL is examinedusing an allergic inflammation animal model (Clin. Immunol.Immunopathol., 75: 75 (1995)). Also, when induced Th2 cells in mice areadministered into the vein and nasal cavity, an antigen-specificasthmatoid inflammatory symptom is induced in the lungs (J. Exp. Med.,186: 1737 (1997), J. Immunol., 160: 1378 (1998)) and causes eosinophilia(J. Immunol., 161: 3128 (1998)). Expression of IL-5 is observed in theairway mucous membrane of asthma patients and the lesions of atopicdermatitis patients (J. Clin. Invest., 87: 1541 (1991), J. Exp. Med.,173; 775 (1991)), and the expression level of IL-13 in the mucousmembrane of continuous allergic rhinitis well correlates with theamounts of serum total IgE and antigen-specific IgE (Therapeutics, 32:19 (1998)).

Chemokine is a general term for basic heparin-binding proteins havingleukocyte migration and leukocyte activation functions, and classifiedinto subfamilies of CXC, CC, C and CX₂C depending on the position of thecysteine residue preserved on the primary structure. Up to date, 16kinds of chemokine receptors have been identified (Curr. Opi. Immunol.,11: 626 (1999)), and it has been shown that expression of each chemokinereceptor is different on the surface of each leukocyte such as Th1 cell,Th2 cell or the like (Cell Engineering, 17: 1022 (1998)).

Human CCR4 is a G protein complexed seven transmembrane receptor clonedas K5-5 from a human immature basophilic cell line KU-812, and has theamino acid sequence represented by SEQ ID NO:17. Since the transmembraneregions of CCR4 are considered to be positions 40-67, positions 78-97,positions 113-133, positions 151-175, positions 207-226, positions243-270 and positions 285-308, it is considered that the extracellulardomains are positions 1-39, positions 98-112, positions 176-206 andpositions 271-284 in the amino acid sequence, and that the intracellularregions are positions 68-77, positions 134-150, positions 227-242 andpositions 309-360 (J. Biol. Chem., 270: 19495 (1995)). When cloning wascarried out, it was reported that the ligand of CCR4 is MIP-1α(macrophage inflammatory protein-1α), RANTES (regulated on activationnormal T-cell expressed and secreted) or MCP-1 (monocyte chemotacticprotein) (Biochem. Biophys. Res. Commun., 218: 337 (1996), WO 96/23068).However, thereafter, it has been found that TARC (thymus andactivation-regulated chemokine) produced from stimulated humanperipheral blood mononuclear cells (hereinafter referred to as “PBMC”)and thymus cells (J. Biol. Chem., 271: 21514 (1996)) specifically bindsto CCR4 (J. Biol. Chem., 272: 15036 (1997)). It has been also reportedthat MDC (macrophage-derived chemokine) isolated from macrophage (J.Exp. Med., 185: 1595 (1997)), also known as STCP-1 (stimulated T cellchemotactic protein-1) (J. Biol. Chem., 272: 25229 (1997)), bind to CCR4more strongly than TARC (J. Biol. Chem., 273: 1764 (1998)).

It has been shown that CCR4 is expressed on CD4+Th cells which producecytokine and/or chemokine (J. Biol. Chem., 272: 15036 (1997)), and ithas been reported that CCR4 is expressed selectively on Th2 cells amongCD4+Th cells (J. Exp. Med., 187: 129 (1998), J. Immunol., 161: 5111(1998)). In addition, CCR4+ cells have been found in a group ofeffector/memory T cells (CD4+/CD45RO+), and when CCR4+ cells werestimulated, IL-4 and IL-5 were produced but IFN-γ (was not produced(Int. Immunol., 11: 81 (1999)). Also, it has been reported that CCR4+cells belong to a CLA (cutaneous lymphocyte antigen)-positive and α4β8integrin-negative group among memory T cells, and CCR4 is expressed onmemory T cells related not to gut immunity but to systemic immunity ofthe skin and the like (Nature, 400: 776 (1999)). These results stronglysuggest a possibility that the memory T cells which are activated byinduction of inflammation express CCR4, migrate into the inflammatorysite by ligands, MDC and TARC, and accelerate activation of otherinflammatory cells.

As the current method for treating Th2-mediated immune diseases, thefollowings have been developed: (1) antagonists for cytokine andchemokine such as a humanized anti-IL-5 antibody (SB-240563: Smith KlineBeecham, Sch-55700 (CDP-835): Shehling Plaw/Celltech), a humanized IL-4antibody (U.S. Pat. No. 5,914,110), a soluble chemokine receptor (J.Immunol., 150: 624 (1998)), etc.; (2) cytokine chemokine productioninhibitors such as an IL-5 production inhibitor (Japanese PublishedUnexamined Patent Application No. 53355/96), a retinoid antagonist (WO99/24024), splatast tosilate (IPD-1151T, manufactured by TaihoPharmaceutical), etc.; (3) agents for eosinophil, mast cell and the likeas final inflammatory cells, such as a humanized IL-5 receptor antibody(WO 97/10354), a CC chemokine receptor 3 (CCR3) antagonist (JapanesePublished Unexamined Patent Application No. 147872/99), etc.; (4)inflammatory molecule inhibitors such as a humanized anti-IgE antibody(Am. J. Respir. Crit. Care Med., 157: 1429 (1998)), etc.; and the like.But they inhibit only a part of the elaborate network among cytokine,chemokine and inflammatory cells and are therefore not radical. Anti-CD4antibodies have an ability to control T cells, and have effects onserious steroid-dependent asthma. However, since the CD4 molecule isbroadly expressed in various immune cells, they lack in specificity andhave a drawback of accompanying strong immunosuppressive effect (Int.Arch. Aller. Immunol., 118: 133 (1999)).

Thus, in order to inhibit all of them, it is required to controlspecifically upstream of the problematic allergic reaction, namely Th2cells.

The currently used principal method for treating patients of seriousTh2-mediated immune diseases is steroid administration, but side effectsby steroids cannot be avoided. Also, there are drawbacks that theconditions of each patient return to the original pathology when thesteroid administration is suspended, and that drug resistance isacquired when the steroid is administered for a long time.

To date, no monoclonal antibody which can detect CCR4-expressing cellsand also has cytotoxicity against CCR4-expressing cells has beenestablished. In addition, no therapeutic agent which can inhibitproduction of Th2 cytokine has been known so far.

Although it has been reported that CCR1 is also expressed on leukemia(Blood, 96: 685 (2000)), no therapeutic agent which injures leukemiacells has been known.

It is known in general that when an antibody derived from a non-humananimal, e.g., a mouse antibody, is administered to human, it isrecognized as an foreign substance and induces a human antibody againstthe mouse antibody (human anti-mouse antibody: hereinafter referred toas “HAMA”) in the human body. It is known that the HAMA reacts with theadministered mouse antibody to cause side effects (J. Clin. Oncol., 2:881 (1984), Blood, 65: 1349 (1985), J. Natl. Cancer Inst., 80: 932(1988), Proc. Natl. Acad. Sci. U.S.A., 82: 1242 (1985)), to quickendisappearance of the administered mouse antibody from the body (J. Nucl.Med., 26: 1011 (1985), Blood, 65: 1349 (1985), J. Natl. Cancer Inst.,80: 937 (1988)), and to reduce therapeutic effects of the mouse antibody(J. Immunol., 135: 1530 (1985), Cancer Res., 46: 6489 (1986)).

In order to solve these problems, attempts have been made to convert anantibody derived from a non-human animal into a humanized antibody suchas a human chimeric antibody, a human complementarity determining region(hereinafter referred to as “CDR”)-grafted antibody or the like usinggenetic engineering technique. The human chimeric antibody is anantibody in which its antibody variable region (hereinafter referred toas “V region”) is an antibody derived from a non-human animal and itsconstant region (hereinafter referred to as “C region”) is derived froma human antibody (Proc. Natl. Acad. Sci. U.S.A., 81: 6851 (1984)). Thehuman CDR-grafted antibody is an antibody in which the amino acidsequence of CDR in the V region derived from a non-human animal antibodyis grafted into an appropriate position of a human antibody (Nature,321: 522 (1986)). In comparison with antibodies derived from non-humananimals such as mouse antibodies and the like, these humanizedantibodies have various advantages for clinical applications. Forexample, regarding immunogenicity and stability in blood, it has beenreported that blood half-life of a human chimeric antibody became about6 times as long as a mouse antibody when administered to human (Proc.Natl. Acad. Sci. U.S.A., 86: 4220 (1989)). In the case of a humanCDR-grafted antibody, it has been reported that its immunogenicity wasreduced and its serum half-life became 4 to 5 times as long as a mouseantibody in experiment using a monkey (J. Immunol., 147: 1352 (1991)).Thus, it is expected that the humanized antibodies have less sideeffects and their therapeutic effects continue for a longer time thanantibodies derived from non-human animals. Also, in treatmentparticularly for reducing the number of CCR4-expressing cells, highercytotoxic activity such as complement-dependent cytotoxic activity(hereinafter referred to as “CDC activity”), antibody-dependentcell-mediated cytotoxic activity (hereinafter referred to as “ADCCactivity”) and the like, via the Fc region (the region in and after thehinge region of an antibody heavy chain) of an antibody are importantfor the therapeutic effects. It has been reported that on such cytotoxicactivities, the human antibodies are superior to antibodies derived fromnon-human animals since the Fc region of human antibodies moreefficiently activates human complement components and human effectorcells having Fc receptor on the cell surface such as monocytes,macrophages, NK cells, and the like, than the Fc region of antibodiesderived from non-human animals. For example, it has been reported thattumor cytotoxic activity by human effector cells was increased in ahuman chimeric antibody prepared by converting the Fc region of an mouseantibody for GD2 into the Fc region of a human antibody (J. Immunol.,144: 1382 (1990)), and similar results have also been reported on ahuman CDR-grafted antibody for CAMPATH-1 antigen (Nature, 332: 323(1988)).

These results clearly show that humanized antibodies are preferred toantibodies derived from non-human animals such as mouse antibodies andthe like, as antibodies for clinical applications to human.

Furthermore, according to the recent advances in protein engineering andgenetic engineering, antibody fragments having a smaller molecularweight such as Fab, Fab′, F(ab′)₂, a single chain antibody (hereinafterreferred to as “scFv”) (Science, 242: 423 (1988)), a disulfidestabilized V region fragment (hereinafter referred to as “dsFv”)(Molecular Immunol., 32: 249 (1995)) and the like, can be produced.Since the fragments are smaller in molecular weight than the completeantibody molecules, they are excellent in transitional ability intotarget tissues (Cancer Res., 52: 3402 (1992)). It is considered thatthese fragments derived from humanized antibodies are more desirablethan those derived from antibodies derived from non-human animals suchas mouse antibodies, when used in clinical applications to human.

As described above, diagnostic and therapeutic effects can be expectedfrom humanized antibodies and fragments thereof when used alone, butattempts have been made to further improve the effects by using othermolecules in combination. For example, cytokine can be used as one ofsuch molecules. Cytokine is a general term for various soluble factorswhich control intercellular mutual functions in immune reactions. CDCactivity and ADCC activity, for example, are known as the cytotoxicactivities of antibodies, and ADCC activity is controlled by effectorcells having Fc receptors on the cell surface such as monocytes,macrophages, NK cells and the like (J. Immunol., 138: 1992 (1987)).Since various cytokines activate these effector cells, they can beadministered in combination with an antibody in order to improve ADCCactivity of the antibody and the like.

SUMMARY OF THE INVENTION

Antibodies bind to the corresponding antigen via the CDRs of V regionsof a heavy chain (hereinafter referred to as “H chain”) and a lightchain (hereinafter referred to as “L chain”), and the amino acidsequence of the CDR regulates binding reactivity and binding specificityof the antibody (J. Exp. Med., 132: 211 (1970)). Thus, there is a demandfor an anti-CCR4 antibody which contains CDRs having a novel amino acidsequence and specifically binds to human CCR4 having certain propertiessuch as binding reactivity, cytotoxic activity and the like, which aredifferent from those of known CCR4 antibodies. Furthermore, there is ademand for an antibody which can selectively deplete CCR4-expressing Th2cells as cytokine-producing cells, an antibody which inhibits productionof Th2 cytokine, and a diagnostic agent and therapeutic agent using theantibody. Moreover, there is a demand for a method useful in diagnosingand treating blood cancers such as leukemia which is a disease caused bytumorigenic transformation of hemopoietic cells, and the like.

The present inventors have obtained cDNAs encoding antibody H chain andL chain from hybridoma KM2160 which produces a mouse monoclonal antibodyfor CCR4 belonging to IgG1 class, have found that the V region CDRs havenovel amino acid sequences, and have constructed a humanized antibodyexpression vector by cloning cDNAs encoding H chain V region and L chainV region having the novel CDRs into an animal cell expression vectorhaving cDNAs encoding human antibody H chain C region and human antibodyL chain C region. Anti-CCR4 chimeric antibody KM2760 was expressed andpurified by introducing the expression vector into animal cells. Thepresent invention has been accomplished by confirming that this antibodyspecifically reacts with human CCR4 and reduces the number ofantigen-positive cells through its potent cytotoxic activity and byshowing utility of the antibody in using in the human body.

In addition, the present inventors have accomplished the presentinvention by confirming that a recombinant antibody for CCR4 reacts withleukemia cells, particularly T cell leukemia cells, at a high frequencyand reduces the number of CCR4-positive Leukemia cells through itspotent cytotoxic activity and by showing utility of the antibody indiagnosing and treating blood cancers such as human leukemia and thelike.

The present invention relates to the following (1) to (47).

(1) A recombinant antibody or the antibody fragment thereof whichspecifically reacts with an extracellular domain of human CCR4.

(2) The recombinant antibody or the antibody fragment thereof accordingto (1), wherein the extracellular domain is an extracellular domainselected from the group consisting of positions 1-39, positions 98-112,positions 176-206, and positions 271-284 in the amino acid sequencerepresented by SEQ ID NO:17.

(3) The recombinant antibody or the antibody fragment thereof accordingto (1) or (2), which recognizes an epitope present in positions 2-29 inthe amino acid sequence represented by SEQ ID NO:17.

(4) The recombinant antibody or the antibody fragment thereof accordingto any one of (1) to (3), which specifically reacts with aCCR4-expressing cell.

(5) The recombinant antibody or the antibody fragment thereof accordingto any one of (1) to (4), which has cytotoxic activity against aCCR4-expressing cell.

Examples of the cytotoxic activity include CDC activity, ADCC activity,and the like.

(6) The recombinant antibody or the antibody fragment thereof accordingto (5), wherein the cytotoxic activity against a CCR4-expressing cell ishigher than that of a monoclonal antibody produced by a hybridomaderived from a non-human animal.

The term “cytotoxic activity higher than that of a monoclonal antibodyproduced by a hybridoma derived from a non-human animal” means that acytotoxic activity of the obtained recombinant antibody is higher thanthat of a monoclonal antibody produced by a hybridoma derived from anon-human animal, which specifically reacts with CCR4 used in producinga recombinant antibody that will be described later.

(7) The recombinant antibody or the antibody fragment thereof accordingto (5), wherein the cytotoxic activity is antibody-dependentcell-mediated cytotoxic (ADCC) activity.

(8) The recombinant antibody or the antibody fragment thereof accordingto (7), wherein the antibody-dependent cell-mediated cytotoxic activityis activity of inducing apoptosis of a Th2 cell.

(9) The recombinant antibody or the antibody fragment thereof accordingto any one of (1) to (8), which has activity of depleting a Th2 cell.

(10) The recombinant antibody or the antibody fragment thereof accordingto any one of (1) to (9), which has activity of inhibiting production ofTh2 cytokine.

(11) The recombinant antibody or the antibody fragment thereof accordingto (10), wherein the Th2 cytokine is IL-4, IL-5 or IL-13.

(12) The recombinant antibody according to any one of (1) to (11),wherein the recombinant antibody is selected from a humanized antibodyand a human antibody.

(13) The recombinant antibody according to (12), wherein the humanizedantibody is a human chimeric antibody or a human CDR-grafted antibody.

(14) The recombinant antibody according to any one of (1) to (13), whichbelongs to a human IgG antibody.

(15) The recombinant antibody according to (12), wherein the humanizedantibody comprises:

complementarity determining region (CDR) 1, CDR2 and CDR3 of an antibodyheavy chain (H chain) variable region (V region) having the amino acidsequences represented by SEQ ID NOs:5, 6 and 7, respectively; and

CDR1, CDR2 and CDR3 of an antibody light chain (L chain) V region havingthe amino acid sequences represented by SEQ ID NOs:8, 9 and 10,respectively.

(16) The recombinant antibody according to (13), wherein the humanchimeric antibody comprises:

an antibody heavy chain (H chain) variable region (V region) andantibody light chain (L chain) V region of a monoclonal antibody whichspecifically reacts with CCR4; and

an H chain constant region (C region) and L chain C region of a humanantibody.

(17) The recombinant antibody according to (16), wherein the humanchimeric antibody comprises:

a complementarity determining region (CDR) 1, CDR2 and CDR3 of an Hchain V region having the amino acid sequences represented by SEQ IDNOs:5, 6 and 7, respectively; and

CDR1, CDR2 and CDR3 of an L chain V region having the amino acidsequences represented by SEQ ID NOs:8, 9 and 10, respectively.

(18) The recombinant antibody according to (16), wherein the humanchimeric antibody comprises:

an H chain V region having amino acids 20-138 of the amino acid sequencerepresented by SEQ ID NO:15; and

an L chain V region having amino acids 20-132 of the amino acid sequencerepresented by SEQ ID NO:16.

(19) The recombinant antibody according to (13), wherein the humanchimeric antibody is an antibody KM2760 produced by a transformantKM2760 (FERN BP-7054), and wherein its antibody H chain C region belongsto human IgG1 subclass.

(20) The recombinant antibody according to (13), wherein the humanCDR-grafted antibody comprises:

complementarity determining regions (CDRs) of an antibody heavy chain (Hchain) variable region (V region) and an antibody light chain (L chain)V region of a monoclonal antibody which specifically reacts with CCR4;and

C regions of an H chain and an L chain and a V region framework regionof a human antibody.

(21) The recombinant antibody according to (20), wherein the humanCDR-grafted antibody comprises:

CDR1, CDR2 and CDR3 of an H chair. V region having the amino acidsequences represented by SEQ ID NOs:5, 6 and 7, respectively; and

CDR1, CDR2 and CDR3 of an L chain V region having the amino acidsequences represented by SEQ ID NOs:8, 9 and 10, respectively.

(22) A DNA which encodes the recombinant antibody or the antibodyfragment thereof according to any one of (1) to (21).

(23) A recombinant vector comprising the DNA according to (22) and atandem vector for humanized antibody expression.

(24) A transformant to which the recombinant vector according to (23) isintroduced into a host cell.

(25) The transformant according to (24), wherein the transformant isKM2760 (FERM BP-7054).

(26) A method for producing a recombinant antibody or the antibodyfragment thereof, which comprises culturing the transformant accordingto (24) or (25) to produce and accumulate the recombinant antibody orthe antibody fragment thereof in a culture medium, and recovering therecombinant antibody or the antibody fragment thereof from the culturemedium.

(27) The recombinant antibody according to (12), wherein the humanantibody comprises an antibody heavy chain (H chain) variable region (Vregion) and/or an antibody light chain (L chain) V region.

(28) The recombinant antibody according to (27), wherein complementaritydetermining regions (CDRs) of the H chain V region and L chain V regionof the human antibody comprise amino acid sequences which are the sameas amino acid sequences of CDRs of an H chain V region and an L chain Vregion, respectively, of a monoclonal antibody which specifically reactswith CCR4.

(29) The recombinant antibody according to (28), wherein the humanantibody comprises:

CDR1, CDR2 and CDR3 of an H chain V region having the amino acidsequences represented by SEQ ID NOs:5, 6 and 7, respectively, and

CDR1, CDR2 and CDR3 of an L chain V region having the amino acidsequences represented by SEQ ID NOs:8, 9 and 10, respectively.

(30) The recombinant antibody according to (27), wherein the H chain Vregion and L chain V region of the human antibody comprise amino acidsequences which are the same as amino acid sequences of an H chain Vregion and an L chain V region, respectively, of a monoclonal antibodywhich specifically reacts with CCR4.

(31) The recombinant antibody according to (30), wherein the humanantibody comprises:

an H chain V region having amino acids of positions 20-138 in the aminoacid sequence represented by SEQ ID NO:15; and/or

an L chain V region having amino acids of positions 20-132 in the aminoacid sequence represented by SEQ ID NO:16.

(32) The recombinant antibody according to any one of (27) to (31),wherein the human antibody is an antibody obtained from a human antibodyphage library or a transgenic animal which produces a human antibody.

(33) The antibody fragment according to any one of (1) to (11), which isFab, Fab′, F(ab′)₂, a single stranded antibody, a disulfide stabilized Vregion fragment, or a peptide comprising a complementarity determiningregion (CDR) of an antibody.

(34) The antibody fragment according to (33), which comprises anantibody heavy chain (H chain) variable region (V region) and anantibody light chain (L chain) V region of an antibody.

(35) The antibody fragment according to (34), wherein complementaritydetermining regions (CDRs) of the H chain V region and L chain V regionof the antibody fragment comprise amino acid sequences which are thesame as amino acid sequences of CDRs of an H chain V region and an Lchain V region, respectively, of a monoclonal antibody whichspecifically reacts with CCR4.

(36) The antibody fragment according to (35), which comprises:

CDR1, CDR2 and CDR3 of the H chain V region having the amino acidsequences represented by SEQ ID NOs:5, 6 and 7, respectively; and

CDR1, CDR2 and CDR3 of the L chain V region having the amino acidsequences represented by SEQ ID NOs:8, 9 and 10, respectively.

(37) The antibody fragment according to (34), wherein the H chain Vregion and L chain V region of the antibody fragment comprise amino acidsequences which are the same as amino acid sequences of an H chain Vregion and an L chain V region, respectively, of a monoclonal antibodywhich specifically reacts with CCR4.

(38) The antibody fragment according to (37), which comprises:

an H chain V region having amino acids of positions 20-138 in the aminoacid sequence represented by SEQ ID NO:15; and

an L chain V region having amino acids of positions of 20-132 in theamino acid sequence represented by SEQ ID NO:16.

(39) A recombinant antibody or the antibody fragment, which is therecombinant antibody or the antibody fragment thereof according to anyone of (1) to (21) and (27) to (38) which is chemically or geneticallyconjugated with a radioisotope, a protein or an agent.

(40) A method for immunologically detecting CCR4, which comprises usingthe recombinant antibody or the antibody fragment thereof according toany one of (1) to (21) and (27) to (39).

For example, CCR4 in a sample can be immunologically detected byallowing the recombinant antibody or the antibody fragment thereof tocontact with the sample.

(41) A method for immunologically detecting a cell which expresses CCR4on the cell surface, which comprises using the recombinant antibody orthe antibody fragment thereof according to any one of (1) to (21) and(27) to (39).

For example, a cell which expresses CCR4 on the cell surface can beimmunologically detected by allowing the recombinant antibody or theantibody fragment thereof to contact with the cell.

(42) A method for reducing or depleting a cell which expressed CCR4 onthe cell surface, which comprises using the recombinant antibody or theantibody fragment thereof according to any one of (1) to (21) and (27)to (39).

For example, the cell which expresses CCR4 on the cell surface can bereduced or depleted by administering an effective amount of therecombinant antibody or the antibody fragment thereof to human or ananimal.

(43) A method for inhibiting production of Th2 cytokine, which comprisesusing the recombinant antibody or the antibody fragment thereofaccording to any one of (1) to (21) and (27) to (39).

For example, production of Th2 cytokine can be inhibited byadministering an effective amount of the recombinant antibody or theantibody fragment thereof to human or an animal.

(44) A medicament comprising, as an active ingredient, the recombinantantibody or the antibody fragment thereof according to any one of (1) to(21) and (27) to (39).

(45) A therapeutic or diagnostic agent for Th2-mediated immune diseases,comprising, as an active ingredient, the recombinant antibody or theantibody fragment thereof according to any one of (1) to (21) and (27)to (39).

For example, a Th2-mediated immune disease can be treated byadministering an effective amount of the recombinant antibody or theantibody fragment thereof to human or an animal, and a Th2-mediatedimmune disease can be diagnosed by allowing the recombinant antibody orthe antibody fragment thereof to contact with a sample to be tested.

(46) A therapeutic or diagnostic agent for a blood cancer, comprising,as an active ingredient, the recombinant antibody or the antibodyfragment thereof according to any one of (1) to (21) and (27) to (39).

For example, a blood cancer can be treated by administering an effectiveamount of the recombinant antibody or antibody fragment thereof to humanor an animal, and a blood cancer can be diagnosed by allowing therecombinant antibody or antibody fragment thereof to contact with asample to be tested.

(47) The therapeutic or diagnostic agent according to (46), wherein theblood cancer is leukemia.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a drawing showing reactivity of KM2160 with a compoundaccording to ELISA.

FIG. 2 is a drawing showing construction steps of plasmids pKM2160VH41and pKM2160VL61.

FIG. 3 is a drawing showing construction step of a plasmid pKANTEX2160H.

FIG. 4 is a drawing showing construction step of a plasmid pKANTEX2160.

FIG. 5 is a drawing showing an SDS-PAGE (5 to 20% gradient gel)electrophoresis pattern of purified anti-CCR4 chimeric antibody KM2760.The left side shows a result of electrophoresis carried out undernon-reducing conditions, and the right side under reducing conditions.Lanes 1, 2, 3 and 4 show electrophoresis patterns of high molecularweight markers, KM2760, low molecular weight markers and KM2760,respectively.

FIG. 6 is a drawing showing the activity of a purified anti-CCR4chimeric antibody KM2760 to bind to a CCR4 partial peptide measured bychanging the antibody concentration. The ordinate and abscissa representthe CCR4-binding activity and the antibody concentration, respectively.FIG. 7 is a drawing showing the reactivity of KM2760 withCCR4-high-expressing cells (CCR4/EL-4) measured by an immunofluorescenceactivating cell sorter.

FIG. 8 is a drawing showing the cytotoxicity by ADCC activity forCCR4/EL-4 cells (upper graph) and EL-4 cells (lower graph).

FIG. 9 is a drawing showing the cytotoxicity by ADCC activity for humanPBMC, measured as annexin V positive ratio in 4 fractions havingdifferent staining abilities of CD4 and CD45RA.

FIG. 10 is a drawing showing the effect of inhibiting production ofIL-4, IL-5, IL-13 and IFN-γ from human PBMC.

FIG. 11 is a drawing showing the reactivity of anti-CCR4 chimericantibody KM2760 with human T cell leukemia cell lines.

FIG. 12 is a drawing showing the cytotoxicity by anti-CCR4 chimericantibody KM2760 for human T cell leukemia cell lines, which reactivitywith anti-CCR4 chimeric antibody KM2760 was confirmed.

FIG. 13 is a drawing showing the changes in characteristics with time ina produced amount of IL-4 when an anti-CCR4 chimeric antibody KM2760 wasadministered to Macaca fascicularis.

FIG. 14 is a drawing showing the changes with time in a produced amountof IL-13 when an anti-CCR4 chimeric antibody KM2760 was administered toMacaca fascicularis.

FIG. 15 is a drawing showing the changes in characteristics with time ina produced amount of IL-γ when an anti-CCR4 chimeric antibody KM2760 wasadministered to Macaca fascicularis.

FIG. 16 is a drawing showing the changes in characteristics with time inthe average value of tumor volumes when an anti-CCR4 chimeric antibodyKM2760 was administered to mice to which hCCR4-high-expressingmouse-derived cells, CCR4/EL-4 cells, had been subcutaneously grafted.

FIG. 17 is a drawing showing the ratio of the average value of tumorvolumes in an administered group to the average value of tumor volumesin a non-administered group when an anti-CCR4 chimeric antibody KM2760was administered to mice to which hCCR4-high-expressing mouse-derivedcells, CCR4/EL-4 cells, had been subcutaneously grafted.

FIG. 18 is a drawing showing the changes in characteristics with time inthe average value of tumor volumes when an anti-CCR4 chimeric antibodyKM2760 was administered to mice to which CCR4-expressing human T cellleukemia line CCRF-CEM cells (ATCC CCL-119) had been subcutaneouslygrafted.

DETAILED DESCRIPTION OF THE INVENTION

This application is based on Japanese applications No. 2000-59508 filedon Mar. 3, 2000 and No. 2000-401563 filed on Dec. 28, 2000, the entirecontents of which are incorporated hereinto by reference.

Examples of the Th2-mediated immune diseases in the present inventioninclude acute or chronic airway hypersensitivity or bronchial asthma,atopic skin diseases including atopic dermatitis, allergic rhinitis,pollinosis and the like.

Examples of the cancer in the present invention include blood cancers,and particularly leukemia.

Any recombinant antibody or the antibody fragment thereof according tothe present invention (hereinafter referred to as the “antibody of thepresent invention”) may be used, so long as it can react specificallywith the extracellular domain of human CCR4. A preferred antibody is anantibody which specifically reacts with a domain comprising positions1-39, positions 98-112, positions 176-206 or positions 271-284 of theamino acid sequence represented by SEQ ID NO:17. More preferredantibodies are an antibody comprising CDR1, CDR2 and CDR3 of an H chainV region having the amino acid sequences represented by SEQ ID NOs:5, 6and 7, respectively, and CDR1, CDR2 and CDR3 of an L chair. V regionhaving the amino acid sequences represented by SEQ ID NOs:8, 9 and 10,respectively; and an antibody comprising an H chain V region havingamino acids of positions 20-138 in the amino acid sequence representedby SEQ ID NO:15, and an L chain V region having amino acids of positions20-132 in the amino acid sequence represented by SEQ ID NO:16.Antibodies and antibody fragments in which one or more amino acids aredeleted, added, substituted and/or inserted in these amino acidsequences and which specifically react with CCR4 are also includedwithin the scope of the present invention.

In the present invention, one or more amino acid deletion, substitution,insertion or addition in the amino acid sequence means that one or moreamino acids are deleted, substituted, inserted and/or added to at one orplural positions in the amino acid sequence. The deletion, substitution,insertion and/or addition may be caused in the same amino acid sequencesimultaneously. Also, the amino acid residue substituted, inserted oradded can be natural or non-natural. Examples of the natural amino acidresidue include L-alanine, L-asparagine, L-aspartic acid, L-glutamine,L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine,L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine, and thelike.

Thereinafter, examples of amino acid residues which are substituted witheach other are shown. The amino acid residues in the same group can besubstituted with each other.

Group A:

leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine, cyclohexylalanine;

Group B:

aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid,2-aminoadipic acid, 2-aminosuberic acid;

Group C:

asparagine, glutamine;

Group D:

lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid;

Group E:

proline, 3-hydroxyproline, 4-hydroxyproline;

Group F:

serine, threonine, homoserine;

Group G:

phenylalanine, tyrosine.

Examples of the antibody of the present invention include a humanizedantibody, a human antibody and the antibody fragment thereof asdescribed below.

Examples of the humanized antibody include a human chimeric antibody anda human CDR-grafted antibody.

A human chimeric antibody is an antibody comprising an antibody H chainV region (hereinafter also referred to as “VH”) and an antibody L chainV region (hereinafter also referred to as “VL”) of a non-human animal, ahuman antibody H chain C region (hereinafter also referred to as “CH”)and a human antibody L chain C region (hereinafter also referred to as“CL”). The non-human animal may be any of mouse, rat, hamster, rabbitand the like, so long as a hybridoma can be prepared therefrom.

The human chimeric antibody of the present invention can be produced byobtaining cDNAs encoding VH and VL from a hybridoma which produces amonoclonal antibody which reacts specifically with CCR4, inserting thecDNAs into an animal cell expression vector having genes encoding ahuman antibody CH and a human antibody CL to construct a human chimericantibody expression vector, and introducing the vector into an animalcell to express the antibody.

Any CH of a human chimeric antibody may be used, so long as it belongsto human immunoglobulin (hereinafter referred to as “hIg”), but those ofhIgG class are preferred, and any one of subclasses further belonging tohIgG such as hIgG1, hIgG2, hIgG3 and hIgG4, can be used. Also, any CL ofa human chimeric antibody may be used, so long as it belongs to hIg, andthose of κ class or λ class can be used.

Examples of the anti-CCR4 chimeric antibody include KM2760 in which VHof the antibody comprises an amino acid sequence of positions 20-138 inthe amino acid sequence represented by SEQ ID NO:15, CH of the antibodycomprises the amino acid sequence of hIgG1 subclass, VL of the antibodycomprises an amino acid sequence of positions 20-132 of the amino acidsequence represented by SEQ ID NO:16, and CL of the antibody has theamino acid sequence of human antibody κ class.

A human CDR-grafted antibody is an antibody in which CDR amino acidsequences of VH and VL of an antibody derived from a non-human animalare grafted into appropriate positions of VH and VL of an humanantibody.

The human CDR-grafted antibody of the present invention can be producedby grafting CDR sequences of VH and VL of an antibody which specificallyreacts with CCR4 of a non-human animal into CDR sequences of VH and VLof an optional human antibody to construct cDNAs encoding V regionsobtained, inserting the cDNAs into an animal cell expression vectorhaving genes encoding human antibody CH and human antibody CL toconstruct a human CDR-grafted antibody expression vector, and thenintroducing the expression vector into an animal cell to express theantibody.

Any CH of human CDR-grafted antibody may be used, so long as it belongsto hIg, but those of hIgG class are preferred and any one of subclassesfurther belonging to hIgG such as hIgG1, hIgG2, hIgG3 and hIgG4, can beused. Also, any CL of human CDR-grafted antibody may be used so long asit belongs to hIg, and those of κ class or λ class can be used.

Originally, a human antibody is an antibody naturally existing in thehuman body, but it also includes antibodies obtained from a humanantibody phage library and a human antibody producing transgenic animal,prepared based on the recent advance in genetic engineering, cellengineering and developmental engineering techniques.

The antibody existing in the human body can be obtained, for example, byisolating a human peripheral blood lymphocyte, immortalizing it byinfection with EB virus or the like, followed by cloning, culturing alymphocyte which produces the antibody, and purifying the antibody fromthe culture supernatant.

The human antibody library is a library in which an antibody fragmentsuch as Fab, scFv or the like, is expressed on the surface of a phage byinserting an antibody gene prepared from human B cell into the phagegene. A phage which expresses an antibody fragment having desiredantigen binding activity can be recovered from the library by using thebinding activity to an antigen-immobilized substrate as the index. Theantibody fragment can be further converted into a human antibodymolecule comprising two complete H chains and two complete L chains bygenetic engineering techniques.

A human antibody-producing transgenic animal is an animal in which ahuman antibody gene is introduced into its cell. Specifically, a humanantibody-producing transgenic animal can be produced by introducing ahuman antibody gene into a mouse ES cell, inoculating the ES cell intoan initial stage embryo of other mouse, and developing an animal. Thehuman antibody may be produced and accumulated by obtaining a hybridomaproducing a human antibody according to a hybridoma preparation methodusually carried out in mammals other than human and then culturing thehybridoma to obtain the human antibody in a culture supernatant.

Examples of the antibody fragment include Fab, Fab′, F(ab′)₂, scFv,dsFv, a peptide comprising CDR, and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and antigen binding activity, in which about a half of the N-terminalside of H chain and the entire L chain, among fragments obtained bytreating IgG with a protease, papain (cut at the 224th position aminoacid residue of the H chain), are bound together through a disulfidebond.

The Fab of the present invention can be obtained by treating an antibodywhich specifically reacts with CCR4, with a protease, papain.Alternatively, the Fab can be produced by inserting DNA encoding Fab ofthe antibody into an expression vector for prokaryote or an expressionvector for eukaryote, and introducing the vector into a prokaryote oreukaryote to express the Fab.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and antigen binding activity, which is slightly larger than theFab bound via a disulfide bond of the hinge region, among fragmentsobtained by treating IgG with a protease, pepsin (cut at the 234thposition amino acid residue of the H chain).

The F(ab′)₂ of the present invention can be obtained by treating anantibody which specifically reacts with CCR4, with a protease, pepsin.Alternatively, it can be produced by binding Fab′ described below via athioether bond or a disulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cutting adisulfide bond of the hinge region of the F(ab′)₂.

The Fab′ of the present invention can be obtained by treating theF(ab′)₂ which specifically reacts with CCR4, with a reducing agent,dithiothreitol. Alternatively, the Fab′ can be produced by inserting DNAencoding Fab′ of the antibody into an expression vector for prokaryoteor an expression vector for eukaryote, and introducing the vector into aprokaryote or eukaryote to express the Fab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked using an appropriate peptide linker (hereinafterreferred to as “P”). The VH and VL in the scFv of the present inventionmay be any antibody of the present invention which specifically reactswith CCR4 such as a humanized antibody or a human antibody.

The scFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of an antibody which specifically reacts with CCR4,constructing DNA encoding scFv, inserting the DNA into an expressionvector for prokaryote or an expression vector for eukaryote, and thenintroducing the expression vector into a prokaryote or eukaryote toexpress the scFv.

A dsFV is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residueto be substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith the method shown by Reiter et al. (Protein Engineering, 7: 697(1994)). As the VH and VL contained in the dsFv of the presentinvention, any antibody of the present invention which specificallyreacts with CCR4 such as a humanized antibody or a human antibody, canbe used.

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of an antibody which specifically reacts with CCR4,constructing DNA encoding dsFv, inserting the DNA into an expressionvector for prokaryote or an expression vector for eukaryote, and thenintroducing the expression vector into a prokaryote or eukaryote toexpress the dsFv.

A peptide comprising CDR is constituted by including at least one regionof H chain and L chain CDRs. Plural CDRs can be bound directly or via anappropriate peptide linker.

The peptide comprising CDR of the present invention can be produced byobtaining cDNA encoding VH or VL of an antibody which specificallyreacts with CCR4, constructing DNA encoding CDR, inserting the DNA intoan expression vector for prokaryote or an expression vector foreukaryote, and then by introducing the expression vector into aprokaryote or eukaryote to express the peptide.

The peptide comprising CDR can also be produced by a chemical synthesismethod such as an Fmoc method (fluorenylmethoxycarbonyl method), a tBocmethod (t-butyloxycarbonyl method), or the like.

The antibody of the present invention includes antibody derivatives inwhich a radioisotope, a protein or an agent is chemically or geneticallyconjugated to an antibody which specifically reacts with CCR4 such as ahumanized antibody, a human antibody or the antibody fragment thereof.

The antibody derivatives of the present invention can be produced bychemically conjugating a radioisotope, a protein or a agent to theN-terminal side or C-terminal side of an H chain or an L chain of anantibody or antibody fragment which specifically reacts with CCR4, to anappropriate substituent group or side chain of the antibody or antibodyfragment or to a sugar chain in the antibody or antibody fragment(Antibody Engineering Handbook, edited by Osamu Kanemitsu, published byChijin Shokan (1994)).

Alternatively, it can be genetically produced by linking a DNA encodingan antibody or antibody fragment which specifically reacts with CCR4 toother DNA encoding a protein to be bound, inserting the DNA into anexpression vector, and introducing the expression vector into a hostcell.

Examples of the isotope include ¹³¹I, ¹²⁵I and the like, and they can beconjugated to antibodies by, e.g., a chloramine T method.

As the agent, a low molecular weight compound is preferred. Examplesinclude anticancer agents such as alkylating agents (e.g., nitrogenmustard, cyclophosphamide, etc.), metabolic antagonists (e.g.,5-fluorouracil, methotrexate, etc.), antibiotics (e.g., daunomycin,bleomycin, mitomycin C, daunorubicin, doxorubicin, etc.), plantalkaloids (e.g., vincristine, vinblastine, vindesine, etc.), hormonedrugs (e.g., tamoxifen, dexamethasone, etc.), and the like (ClinicalOncology, edited by Japanese Society of Clinical Oncology, published byCancer and Chemotherapy (1996)); anti-inflammatory agents such assteroid agents (e.g., hydrocortisone, prednisone, etc.), non-steroidaldrugs (e.g., aspirin, indometacin, etc.), immunomodulators (e.g.,aurothiomalate, penicillamine, etc.), immunosuppressing agents (e.g.,cyclophosphamide, azathioprine, etc.), antihistaminic agents (e.g.,chlorpheniramine maleate, clemastine, etc.), and the like (Inflammationand Anti-inflammatory Therapy, Ishiyaku Shuppan (1982)); and the like.Examples of the method for conjugating daunomycin to an antibody includea method in which daunomycin and an amino group of an antibody areconjugated via glutaraldehyde, a method in which an amino group ofdaunomycin and a carboxyl group of an antibody are conjugated via awater-soluble carbodiimide, and the like.

As the protein, cytokine which activates immune cells is preferred asthe protein. Examples include human interleukin 2 (hereinafter referredto as “hIL-2”), human granulocyte macrophage colony-stimulating factor(hereinafter referred to as “hGM-CSF”), human macrophagecolony-stimulating factor (hereinafter referred to as “hM-CSF”), humaninterleukin 12 (hereinafter referred to as “hIL-12”), and the like.Also, in order to inhibit cancer cells directly, a toxin such as ricin,diphtheria toxin and the like, can be used. For example, a fusionantibody with a protein can be produced by linking a cDNA encoding anantibody or antibody fragment to other cDNA encoding the protein,constructing DNA encoding the fusion antibody, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing it into a prokaryote or eukaryote to express thefusion antibody.

Methods for producing a human chimeric antibody which reactsspecifically with CCR4 and has novel amino acid sequences in the Vregions of H chain and L chain are explained below based on examples.

1. Production of Anti-CCR4 Monoclonal Antibody Produced by Hybridoma

(1) Preparation of Antigen

A recombinant CCR4 protein is obtained by introducing an expressionvector containing cDNA encoding CCR4 into a host cell such asEscherichia coli, yeast, an insect cell, an animal cell or the like.Alternatively, a cultured tumor cell which expresses CCR4, a CCR4protein purified from the cell or a synthetic peptide having a CCR4partial sequence can be used as the antigen.

A partial protein sequence having approximately 5 to 30 residues isselected as a partial peptide for an antigen. In order to obtain anantibody which recognizes the protein having an unmodified naturalstructure, it is necessary to select a partial sequence existing on thesurface of the three-dimensional structure of the protein as the antigenpeptide. The part existing on the surface of the three-dimensionalstructure of the protein can be expected by estimating a partialsequence having high hydrophilicity using a commercially availableprotein sequence analyzing software such as Genetyx Mac or the like. Ingeneral, portions having low hydrophilicity are mostly present insidethe three-dimensional protein structure, while portions having highhydrophilicity are mostly present on the protein surface. Also, theN-terminal and C-terminal of a protein are present on the proteinsurface in many cases. However, a partial peptide selected in thismethod does not always function as an antigen which establishes thetarget antibody.

In order to crosslink the partial peptide with the protein, a cysteineresidue is added to the terminal region of the partial peptide. When aninternal sequence of the protein is selected, N-terminal and C-terminalof the peptide are acetylated and amidated, respectively, if necessary.

The partial peptide can be synthesized by a usual liquid phase or solidphase peptide synthesis method, the combined method thereof or themodified method thereof (The Peptides, Analysis, Synthesis, Biology,vol. 1, edited by Erhard Gross and Johannes Meinhofer, Academic Press(1979), vol. 2 (1980), vol. 3 (1981); Fundamentals and Experiments onPeptide Synthesis, Nobuo Izumiya, Maruzen (1985); Development ofDrugs—Second Series, vol. 14, Peptide Synthesis, edited by HaruakiYajima, Hirokawa Shoten (1991); International Journal of Peptide ProteinResearch, 35: 161 (1990)).

Also, an automatic peptide synthesizer can be used. A peptide can besynthesized by a peptide synthesizer using amino acids with appropriateprotected side chains such as Nα-Fmoc-amino acid, Nα-Boc-amino acid, andthe like, on a commercially available peptide synthesizer, for example,a peptide synthesizer manufactured by Shimadzu, a peptide synthesizermanufactured by Applied Biosystems, Inc., USA (hereinafter referred toas “ABI”), a peptide synthesizer manufactured by Advanced ChemTech Inc.,USA (hereinafter referred to as “ACT”), or the like, according to thesynthesis program of each synthesizer.

Protected amino acids and carrier resins as starting materials can bepurchased from ABI, Shimadzu, Kokusan Kagaku, Nova Biochem, WatanabeKagaku, ACT, Peptide Institute or the like. Also, the protected aminoacids, protected organic acids and protected organic amines as startingmaterials can be synthesized by known synthesis methods or modifiedmethods thereof (The Peptides, Analysis, Synthesis, Biology, vol. 1,edited by Erhard Gross and Johannes Meinhofer, Academic Press (1979),vol. 2 (1980), vol. 3 (1981); Fundamentals and Experiments on PeptideSynthesis, Nobuo Izumiya, Maruzen (1985); Development of Drugs—SecondSeries, vol. 14, Peptide Synthesis, edited by Haruaki Yajima, HirokawaShoten (1991); International Journal of Peptide Protein Research, 35:161 (1990)).

(2) Immunization of Animal and Preparation of Antibody-Producing Cell

Any animal such as mice, rats, hamsters, rabbits and the like, can beused in the immunization, so long as a hybridoma can be produced. Anexample using mice and rats is described below.

A 3- to 20-weeks-old mouse or rat is immunized with the antigen preparedin the above 1(1), and antibody-producing cells are collected from thespleen, lymph node or peripheral blood of the animal. The immunizationis carried out by administering the antigen to the animal several timesthrough subcutaneous, intravenous or intraperitoneal injection togetherwith an appropriate adjuvant. Examples of the adjuvant include acomplete Freund's adjuvant, a combination of aluminum hydroxide gel withpertussis vaccine, and the like. When the antigen is a partial peptide,a conjugate is produced with a carrier protein such as BSA (bovine serumalbumin), KLH (keyhole limpet hemocyanin) or the like, which is used asthe antigen. Three to seven days after each administration, a bloodsample is collected from the fundus of the eye or caudal vein of theanimal, the reactivity with the antigen used, CCR4, is tested, forexample, by enzyme immunoassay (Enzyme-linked Immunosorbent Assay(ELISA), published by Igaku Shoin (1976)), and then a mouse or ratshowing a sufficient antibody titer in their sera is used as the supplysource of antibody-producing cells. On the 3rd to 7th days after finaladministration of the antigen, the spleen is excised from the immunizedmouse or rat to carry out fusion of the spleen cells with myeloma cellsaccording to the known method (Antibodies—A Laboratory Manual, ColdSpring Harbor Laboratory (1988)).

(3) Preparation of Myeloma Cell

Any myeloma cell can be used, so long as it proliferates in vitro.Examples include established cell lines obtained from mouse such as8-azaguanine-resistant mouse (BALB/c) myeloma cell line P3-X63Ag8-U1(P3-U1) (Europ. J. Immunol, 6: 511 (1976)), SP2/0-Ag14 (SP-2) (Nature,276: 259 (1978)), P3-X63-Ag8653 (653) (J. Immunol., 123: 1548 (1979)),P3-X63-Ag8 (X63) (Nature, 256: 495 (1975)), and the like. These celllines are cultured and subcultured according to the known method(Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988))and 2×10⁷ or more of the cells are secured until cell fusion.

(4) Cell Fusion

The above-obtained antibody-producing cells are washed, a cellaggregating medium such as polyethylene glycol-1000 (PEG-1000) or thelike, was added thereto to fuse the cells, and the cells are suspendedin the medium. For washing the cells, MEM medium, PBS (1.83 g ofdisodium hydrogen phosphate, 0.21 g of potassium dihydrogen phosphate,7.65 q of sodium chloride, 1 liter of distilled water, pH 7.2) or thelike can be used. Also, in order to obtain the target fused cellsselectively, HAT medium {normal medium (a medium prepared by addingglutamine (1.5 mM), 2-mercaptoethanol (5×10⁻⁵ M), gentamicin (10 μg/ml)and fetal calf serum (FCS) (10%, manufactured by CSL) to RPHT-1640medium further supplemented with hypoxanthine (10⁻⁴ M), thymidine(1.5×10⁻⁵ M) and aminopterin (4×10⁻⁷ M)} can be used as the medium forsuspending the fused cells.

After the culturing, a portion of the culture supernatant is sampled anda sample which reacts with an antigen protein but does not react to anon-antigen protein is selected by enzyme immunoassay. Thereafter,cloning is carried out by a limiting dilution method, and a hybridomawhich shows a stably high antibody titer is selected as the monoclonalantibody-producing hybridoma.

(5) Selection of Hybridoma Producing Anti-CCR4 Monoclonal Antibody

A hybridoma producing an anti-CCR4 monoclonal antibody is selected bythe assay described below according to the method described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory (1988) orthe like. According to the assay, the binding activity of the anti-CCR4human antibody contained in a culture supernatant of a transformantproducing the anti-CCR4 human chimeric antibody described below orantibody fragment, or all purified anti-CCR4 antibodies can be measured.

Enzyme Immunoassay:

An antigen is coated on a 96-well ELISA plate. A reaction is carried outusing a hybridoma culture supernatant or a purified antibody obtained inthe above method as a first antibody.

After the reaction of the first antibody, the plate is washed and asecond antibody is added thereto.

The second antibody is obtained by Labeling an antibody which canrecognize immunoglobulin of the first antibody with biotin, an enzyme, achemiluminescent substance, a radioactive compound or the like. If amouse is used for the production of the hybridoma, an antibody which canrecognize mouse immunoglobulin is used as a second antibody.

After the reaction, a reaction suitable for the substance used forlabeling the second antibody is carried out to select a hybridomaproducing a monoclonal antibody which specifically reacts with theantigen.

Examples of the hybridoma include hybridoma KM2160.

(6) Purification of Monoclonal Antibody

The hybridoma cells producing an anti-CCR4 monoclonal antibody obtainedin the above 1(4) are administered by intraperitoneal injection into 8-to 10-weeks-old mice or nude mice treated with pristane (0.5 ml of2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by feeding for 2 weeks) at a dose of 2×10⁷ to5×10⁶ cells/animal. The hybridoma causes ascites tumor in 10 to 21 days.The ascitic fluid is collected from the mice or nude mice, centrifuged,subjected to salting out with 40 to 50% saturated ammonium sulfate or tocaprylic acid precipitation, and then passed through a DEAE-Sepharosecolumn, protein A column or Cellulofine GSL 2000 (manufactured bySeikagaku Corporation) to collect an IgG or IgM fraction as a purifiedmonoclonal antibody.

The subclass of the purified monoclonal antibody can be determined usinga mouse monoclonal antibody typing kit or a rat monoclonal antibodytyping kit. The amount of the protein can be determined by the Lowrymethod or by absorbance at 280 nm.

The subclass of an antibody means isotypes within the class such asIgG1, IgG2a, IgG2b and IgG3 in the case of mouse, and IgG1, IgG2, IgG3and IgG4 in the case of human.

The mouse IgG2a, IgG2b and IgG3 and human IgG1 and IgG3 types haverelatively high cytotoxic activity such as CDC activity, ADCC activityand the like, so that they are useful in applying to medical treatments.

2. Production of Humanized Antibody

(1) Construction of Humanized Antibody Expression Vector

A humanized antibody expression vector is an expression vector foranimal cell into which genes encoding an H chain C region and an L chainC region of a human antibody have been inserted, and is constructed bycloning each of the H chain C region and L chain C region of a humanantibody into an expression vector for animal cell.

The C region of a human antibody may be an H chain C region and an Lchain C region of any human antibody. Examples include a C regionbelonging to IgG1 subclass of an H chain of a human antibody(hereinafter referred to as “hCγ1”), a C region belonging to κclass ofan L chain of a human antibody (hereinafter referred to as “hCκ”), andthe like. As the gene encoding the H chain C region and L chain C regionof a human antibody, a chromosomal DNA comprising an exon and an intronor cDNA can be used.

As the expression vector for animal cell, any expression vector can beused, so long as a C region of a human antibody can be insertedthereinto and expressed therein. Examples include pAGE107(Cytotechnology, 3: 133 (1990)), pAGE103 (J. Biochem., 101: 1307(1987)), pHSG274 (Gene, 27: 223 (1984)), pKCR (Proc. Natl. Acad. Sci.USA, 78: 1527 (1981)), pSGI βd2-4 (Cytotechnology, 4: 173 (1990)), andthe like. Examples of a promoter and enhancer used for an expressionvector for animal cell include an SV40 early promoter and enhancer (J.Biochem., 101: 1307 (1987)), a Moloney mouse leukemia virus LTR promoterand enhancer (Biochem. Biophys. Res. Comun., 149: 960 (1987)), animmunoglobulin H chain promoter (Cell, 41: 479 (1985)) and enhancer(Cell, 33: 717 (1983)), and the like.

The humanized antibody expression vector may be either of a type inwhich a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In respect of easiness ofconstruction of a humanized antibody expression vector, easiness ofintroduction into animal cells, and balance between the expressionamounts of antibody H and L chains in animal cells, a tandem type of thehumanized antibody expression vector is more preferred (J. Immunol.Methods, 167: 27L (1994)). Examples of the tandem type of the humanizedantibody expression vector include pKANTEX93 (WO 97/10354), pEE18(HYBRIDOMA, 17: 559 (1998)), and the like.

The constructed humanized antibody expression vector can be used forexpression of a human chimeric antibody and a human CDR-grafted antibodyin animal cells.

(2) Preparation of cDNA Encoding V Region of Antibody Derived fromNon-Human Animal and Analysis of Amino Acid Sequence

cDNAs encoding the H chain V region and L chain V region of an antibodyderived from an non-human animal such as a mouse antibody are obtainedas follows.

mRNA is extracted from hybridoma cells producing a mouse antibody or thelike to synthesize cDNA. The synthesized cDNA is inserted into a vectorsuch as a phage, a plasmid or the like, to prepare a cDNA library. Eachof a recombinant phage or recombinant plasmid containing cDNA encodingan H chain V region and a recombinant phage or recombinant plasmidcontaining cDNA encoding an L chain V region is isolated from thelibrary using a part of the C region or V region of a mouse antibody asthe probe. The full nucleotide sequences of the H chain V region and Lchain V region of the mouse antibody of interest on the recombinantphage or recombinant plasmid are determined, and the full amino acidsequences of the H chain V region and L chain V region are deduced fromthe nucleotide sequences.

The non-human animal may be any animal such as mouse, rat, hamster,rabbit or the like, so long as a hybridoma cell can be producedtherefrom.

Examples of the method for preparing total RNA from a hybridoma cellinclude a guanidine thiocyanate-cesium trifluoroacetate method (Methodsin Enzymol., 154: 3 (1987)) and the like. Examples of the method forpreparing mRNA from total RNA include an oligo (dT) immobilizedcellulose column method (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Lab. Press, New York (1989)) and the like. Also, examplesof a kit for preparing mRNA from a hybridoma cell include Fast TrackmRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNAPurification Kit (manufactured by Pharmacia) and the like.

Examples of the method for synthesizing cDNA and preparing a cDNAlibrary include known methods (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Lab. Press, New York (1989); Current Protocols inMolecular Biology, Supplement 1-34); a method using a commerciallyavailable kit such as Super Script™ Plasmid System for cDNA Synthesisand Plasmid Cloning (manufactured by GIBCO BRL), ZAP-cDNA Kit(manufactured by Stratagene), etc.; and the like.

The vector into which the cDNA synthesized using mRNA extracted from ahybridoma cell as the template is inserted for preparing a cDNA librarymay be any vector, so long as the cDNA can be inserted. Examples includeZAP Express (Strategies, 5: 58 (1992)), pBluescript II SK(+) (NucleicAcids Research, 17: 9494 (1989)), λzapII (manufactured by Stratagene),λgt10 and λgt11 (DNA Cloning: A Practical Approach, 1,49 (1985)), LambdaBlueMid (manufactured by Clontech), λExCell and pT7T3 18U (manufacturedby Pharmacia), pcD2 (Mol. Cell. Biol., 3: 280 (1983)), pUC18 (Gene, 33:103 (1985)), and the like.

Any E. coli for introducing the cDNA library constructed by a phage orplasmid vector may be used, so long as the cDNA library can beintroduced, expressed and maintained. Examples include XL1-Blue MRF′(Strategies, 5: 81 (1992)), C600 (Genetics, 39: 440 (1954)), Y1088 andY1090 (Science, 222: 778 (1983)), NM522 (J. Mol. Biol., 166: 1 (1983)),K802 (J. Mol. Biol., 16: 118 (1966)), JM105 (Gene, 38: 275 (1985)), andthe like.

A colony hybridization or plaque hybridization method using an isotope-or fluorescence-labeled probe may be used for selecting cDNA clonesencoding an H chain V region and an L chain V region of an antibodyderived from a non-human animal in the cDNA library (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Lab. Press, New York (1989)).Also, the cDNAs encoding an H chain V region and an L chain V region canbe prepared through polymerase chain reaction (hereinafter referred toas “PCR”; Molecular Cloning: A Laboratory Manual, Cold Spring HarborLab. Press, New York, 1989; Current Protocols in Molecular Biology,Supplement 1-34) by preparing primers and using cDNA prepared from mRNAor a cDNA library as the template.

The nucleotide sequence of the cDNA can be determined by digesting thecDNA selected by the above method with appropriate restriction enzymesand the like, cloning the fragments into a plasmid such as pBluescriptSK(−) (manufactured by Stratagene) or the like, carrying out thereaction by a usually used nucleotide analyzing method such as thedideoxy method of Sanger, F. et al. (Proc. Natl. Acad. Sci. USA, 74:5463 (1977)) or the like, and then analyzing the sequence using anautomatic nucleotide sequence analyzer such as A.L.F. DNA sequencer(manufactured by Pharmacia) or the like.

Whether the obtained cDNAs encode the full amino acid sequences of the Hchain V region and L chain V region of the antibody containing asecretory signal sequence can be confirmed by estimating the full aminoacid sequences of the H chain V region and L chain V region from thedetermined nucleotide sequence and comparing them with full amino acidsequences of the H chain V region and L chain V region of knownantibodies (Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)). The length of the secretory signalsequence and N-terminal amino acid sequence can be deduced by comparingthe full amino acid sequences of the H chain V region and L chain Vregion of the antibody comprising a secretory signal sequence with fullamino acid sequences of the H chain V region and L chain V region ofknown antibodies (Sequences of Proteins of Immunological Interest, USDept. Health and Human Services (1991)), and the subgroup to which theybelong can be known. Furthermore, the amino acid sequence of each ofCDRs of the H chain V region and L chain V region can be found bycomparing the obtained amino acid sequences with amino acid sequences ofthe H chain V region and L chain V region of known antibodies (Sequencesof Proteins of Immunological Interest, US Dept. Health and HumanServices (1991)).

Moreover, the novelty of the sequence can be examined by carrying out ahomology search with sequences in any database, for example, SWISS-PROT,PIR-Protein or the like using the full amino acid sequences of the Hchain V region and L chain V region, for example, according to the BLASTmethod (J. Mol. Biol., 215: 403 (1990)) or the like.

(3) Construction of Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed bycloning cDNAs encoding an H chain V region and an L chain V region of anantibody derived from a non-human animal in a region upstream of genesencoding an H chain C region and an L chain C region of a human antibodyon the humanized antibody expression vector as described in the above2(1). For example, each of cDNAs encoding an H chain V region and an Lchain V region of an antibody derived from a non-human animal is linkedwith a synthesized DNA comprising nucleotide sequences at the 3′ ends ofan H chain V region and an L chain V region of an antibody derived froma non-human animal and nucleotide sequences at the 5′ ends of an H chainC region and an L chain C region of a human antibody and having arecognition sequence of an appropriate restriction enzyme at both ends,and each cDNA is cloned so that it is appropriately expressed inupstream of genes encoding the H chain C region and L chain C region ofthe humanized antibody expression vector as described in the above 2(1)to thereby construct a human chimeric antibody expression vector. Also,cDNAs of the H chain V region and L chain V region of an antibodyderived from a non-human animal are amplified by PCR using a syntheticDNA having a recognition sequence of an appropriate restriction enzymeat both ends, and each can be cloned into the humanized antibodyexpression vector as described in the above 2(1).

(4) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

cDNAs encoding an H chain V region and an L chain V region of a humanCDR-grafted antibody can be obtained as follows. First, amino acidsequences of FRs in an H chain V region and an L chain V region of ahuman antibody to which amino acid sequences of CDRs in an H chain Vregion and an L chain V region of an antibody derived from a non-humananimal antibody are grafted are selected. Any amino acid sequences ofFRs in an H chain V region and an L chain V region of a human antibodycan be used, so long as they are derived from human. Examples includeamino acid sequences of FRs in an H chain V region and an L chain Vregion of human antibodies registered in database such as Protein DataBank or the like, and amino acid sequences common to subgroups of FRs inthe H chain V region and L chain V region of human antibodies (Sequencesof Proteins of Immunological Interest, US Dept. Health and HumanServices (1991)), and the like. In order to produce a human CDR-graftedantibody having potent activity, amino acid sequences having highhomology (at least 60% or more) with amino acid sequence of FRs of an Hchain V region and an L chain V region of a target antibody derived froma non-human animal is preferably selected. Then, amino acid sequences ofCDRs of an H chain V region and an L chain V region of the antibodyderived from a non-human animal are grafted to the selected amino acidsequences of FRs of an H chain V region and an L chain V region of ahuman antibody to design amino acid sequences of the H chain V regionand L chain V region of a human CDR-grafted antibody. The designed aminoacid sequences are converted to DNA sequences by considering thefrequency of codon usage found in nucleotide sequences of genes ofantibodies (Sequence of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)), and the DNA sequences encoding theamino acid sequences of the H chain V region and L chain V region of ahuman CDR-grafted antibody are designed. Several synthetic DNAs having alength of about 100 nucleotides are synthesized, and PCR is carried outusing them. In this case, it is preferred in each of the H chain and theL chain that 6 synthetic DNAs are designed in view of the reactionefficiency of PCR and the lengths of DNAs which can be synthesized.

Furthermore, they can be easily cloned into the humanized antibodyexpression vector constructed in the above 2(1) by introducing therecognition sequence of an appropriate restriction enzyme to the 5′ endof the synthetic DNAs present on the both ends. After the PCR, anamplified product is cloned into a plasmid such as pBluescript SK (−)(manufactured by Stratagene) or the like, and the nucleotide sequencesare determined according to the method described in the above 2(2) toobtain a plasmid having DNA sequences encoding the H chain V region andL chain V region of a designed human CDR-grafted antibody.

(5) Modification of Amino Acid Sequence of V Region of Human CDR-GraftedAntibody

It is known that when a human CDR-grafted antibody is produced by simplygrafting only CDRs in an H chain V region and an L chain V region of anantibody derived from a non-human animal in FRs of an H chain V regionand an L chain V region of a human antibody, its antigen-bindingactivity is lower than that of the original antibody derived from anon-human animal (BIO/TECHNOLOGY, 9: 266 (1991)). As the reason, it isconsidered that several amino acid residues in not only CDRs but alsoFRs directly or indirectly relate to antigen-binding activity in the Hchain V region and the L chain V region of the original antibody derivedfrom a non-human animal, and that they are changed to different aminoacid residues of different FRs in the H chain V region and the L chain Vregion of a human antibody. In order to solve the problem, in humanCDR-grafted antibodies, among the amino acid sequences of FRs in an Hchain V region and an L chain v region of a human antibody, an aminoacid residue which directly relates to binding to an antigen, or anamino acid residue which indirectly relates to binding to an antigen byinteracting with an amino acid residue in CDR or by maintaining thethree-dimensional structure of an antibody is identified and modified toan amino acid residue which is found in the original non-human animalantibody to thereby increase the antigen binding activity which has beendecreased (BIO/TECHNOLOGY, 9: 266 (1991)). In the production of a humanCDR-grafted antibody, how to efficiently identify the amino acidresidues relating to the antigen binding activity in FR is mostimportant, so that the three-dimensional structure of an antibody isconstructed and analyzed by X-ray crystallography (J. Mol. Biol., 112:535 (1977)), computer-modeling (Protein Engineering, 7: 1501 (1994)) orthe like. Although the information of the three-dimensional structure ofantibodies has been useful in the production of a human CDR-graftedantibody, no method for producing a human CDR-grafted antibody which canbe applied to any antibodies has been established yet. Therefore,various attempts must be currently be necessary, for example, severalmodified antibodies of each antibody are produced and the relationshipbetween each of the modified antibodies and its antibody bindingactivity is examined.

The modification of the selected amino acid sequence of FR in the Hchain V region and the L chain V region of a human antibody can beaccomplished using various synthetic DNA for modification according toPCR as described in the above 2(4). With regard to the amplified productobtained by the PCR, the nucleotide sequence is determined according tothe method as described in the above 2(2) so that whether the objectivemodification has been carried out is confirmed.

(6) Construction of Human CDR-Grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed bycloning cDNAs encoding the H chain V region and the L chain V region ofthe human CDR-grafted antibody constructed in the above 2(4) and 2(5)into upstream of the genes encoding the H chain C region and the L chainC region of the human antibody in the humanized antibody expressionvector as described in the above 2(1).

For example, when recognition sites for an appropriate restrictionenzymes are introduced to the 5′-terminal of synthetic DNAs positionedat both ends among synthetic DNAs used in the construction of the Hchain V region and the L chain V region of the human CDR-graftedantibody in the above 2(4) and 2(5), cloning can be carried out so thatthey are expressed in an appropriate form in upstream of genes encodingthe H chain C region and the L chain C region of the human antibody inthe humanized antibody expression vector as described in the above 2(1).

(7) Transient Expression of Humanized Antibodies

In order to efficiently evaluate the antigen binding activity of varioushumanized antibodies produced, the humanized antibodies can be expressedtransiently using the humanized antibody expression vector as describedin the above 2(3) and 2(6) or the modified expression vector thereof.Any cell can be used as a host cell, so long as the host cell canexpress a humanized antibody. Generally, COS-7 cell (ATCC CRL1651) isused in view of its high expression amount (Methods in Nucleic AcidsRes., CRC Press, p. 283 (1991)). Examples of the method for introducingthe expression vector into COS-7 cell include a DEAE-dextran method(Methods in Nucleic Acids Res., CRC Press, p. 283 (1991)), a lipofectionmethod (Proc. Natl. Acad. Sci. USA, 84: 7413 (1987)), and the like.

After introduction of the vector, the expression amount and antigenbinding activity of the humanized antibody in the culture supernatantcan be determined by the enzyme immunoassay ((ELISA); Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988),Monoclonal Antibodies: Principles and Practice, Academic Press Limited(1996)), and the like.

(8) Stable Expression of Humanized Antibody

A transformant which produces a humanized antibody stably can beobtained by introducing into an appropriate host cell the humanizedantibody expression vector described in the above 2(3) and 2(6).

Examples of the method for introducing the expression vector into a hostcell include electroporation (Japanese Published Unexamined PatentApplication No. 257891/90, Cytotechnology, 3: 133 (1990)) and the like.

Any cell can be used as the host cell into which the humanized antibodyexpression vector is to be introduced, provided that it can express ahumanized antibody. Examples include mouse SP2/0-Ag14 cell (ATCCCRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which adihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) isdetective (Proc. Natl. Acad. Sci. U.S.A., 77: 4216 (1980)), ratYB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as“YB2/0 cell”), and the like.

A host cell for producing an antibody having antibody-dependentcell-mediated cytotoxic activity is preferably a cell having a lowenzyme activity or no enzyme activity relating to a reaction in whichfucose is added to N-acetylglucosamine bound to the Fc region of theantibody.

Examples of an enzyme for adding fucose to N-acetylglucosamine includeenzymes relating to α1,6-bond such as α1,6-fucosyltransferase, enzymesrelating to biosynthesis of GDP-fucose, e.g., GDP-mannose4,6-dehydratase, GDP-β-L-fucosepyrophosphorylase, focokinase, and thelike. Accordingly, cells obtained by subjecting cells used as the hostcell to artificial mutation in which the gene of the enzyme is deletedor the gene is mutated to thereby reduce or delete the enzyme activitycan also be used as the host cell.

As the host cell, any cell of bacteria, yeast, animal cells, insectcells, plant cells and the like can be used, so long as the recombinantantibody can be produced therein. Animal cells are preferred, andexamples include YB2/0 cells, mouse myeloma cells such as NS0 cell andSP2/0 cell, Chinese hamster ovarian cells such as CHO/dhfr cell andCHO/DG44 cell, monkey cells such as COS cell, human myeloma cells suchas Namalwa cell, and the like.

An antibody containing the sugar chain bound to the Fc region of theantibody having a high N-glucoside binding sugar chain content in whichfucose does not bind to N-acetylglucosamine can be obtained by using thehost cell. The antibody shows higher antibody-dependent cell-mediatedcytotoxic activity against CCR4-expressing cells than a monoclonalantibody produced by a hybridoma derived from a non-human animal.

After introduction of the expression vector, transformants which expressa humanized antibody stably are selected in accordance with the methoddisclosed in Japanese Published Unexamined Patent Application No.257891/90, by culturing in a medium for animal cell culture containingan agent such as G418 sulfate (hereinafter referred to as “G418”,manufactured by Sigma) or the like. Examples of the medium for animalcell culture include PRMI1640 medium (manufactured by NissuiPharmaceutical), GIT medium (manufactured by Nissui Pharmaceutical),EX-CELL302 medium (manufactured by JRH), IMDM medium (manufactured byGIBCO BRL), Hybridoma-SFM medium (manufactured by GIBCO BRL), mediaobtained by adding various additives such as FCS to these media, and thelike. The humanized antibody can be produced and accumulated in aculture medium by culturing the selected transformants in a medium. Theexpression amount and antigen binding activity of the humanized antibodyin the culture supernatant can be measured by ELISA or the like. Also,in the transformant, the expression amount of the humanized antibody canbe increased by using dhfr amplification system or the like according tothe method disclosed in Japanese Published Unexamined Patent ApplicationNo. 257891/90.

The humanized antibody can be purified from the culture supernatant ofthe transformant by using a protein A column (Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Chapter 8 (1988), MonoclonalAntibodies: Principles and Practice, Academic Press Limited (1996)). Anyother conventional methods for protein purification can be used. Forexample, the humanized antibody can be purified by a combination of gelfiltration, ion-exchange chromatography, ultrafiltration and the like.The molecular weight of the H chain or the L chain of the purifiedhumanized antibody or the antibody molecule as a whole is determined bypolyacrylamide gel electrophoresis (hereinafter referred to as“SDS-PAGE”) [Nature, 227: 680 (1970)], Western blotting (Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 12 (1988),Monoclonal Antibodies: Principles and Practice, Academic Press Limited(1996)), and the like.

(9) Evaluation of Activity of Humanized Antibody

The binding reactivity to an antigen and the binding activity to aCCR4-expressing cell line of the purified humanized antibody can bedetermined by ELISA, an immunofluorescent method (Cancer ImmunoImmunother., 36: 373 (1993)). The cytotoxic activity against an antigenpositive culture cell line can be evaluated by measuring the CDCactivity, the ADCC activity or the like (Cancer Immunol. Immunother.,36: 373 (1993)).

3. Method for Detecting and Determining CCR4 Using Anti-CCR4-Antibody

The present invention relates to a method for immunologically detectingand determining CCR4 or a cell expressing CCR4 on the surface thereofusing the antibody of the present invention.

The method for immunologically detecting and determining CCR4 or a cellexpressing CCR4 on the surface thereof using the antibody of the presentinvention include an immunofluorescent method, an enzyme-linkedimmunosorbent assay (ELISA), a radioactive material labeled immunoassay(RIA), an immunohitsochemical staining method such as an immunocytestaining method, an immunotissue staining method, or the like (ABCmethod, CSA method, etc.), the above enzyme immunoassay, a sandwichELISA (Monoclonal Antibody Experiment Manual (published by KodanshaScientific, 1987), Second Series Biochemical Experiment Course, Vol. 5,Immunobiochemistry Research Method, published by Tokyo Kagaku Dojin(1986)).

The immunofluorescent method comprises reacting a separated cell,tissue, or the like with the antibody of the present invention, reactingthe reactant with an anti-immunoglobulin antibody or binding fragmentlabeled with a fluorescence substance such as fluorescein isothiocyanate(FITC) or the like, and then measuring the fluorescence substance with aflow cytometer.

The enzyme-linked immunosorbent assay (ELISA) comprises reacting aseparated cell or crushing solution thereof, tissue or crushing solutionthereof, cell culture supernatant, serum, preural fluid, ascites fluid,ocular fluid or the like with the antibody of the present invention,reacting the reactant with an anti-immunoglobulin antibody or bindingfragment labeled with an enzyme such as peroxydase, biotin, or the like,and then measuring the resultant developed dye with an absorptionphotometer.

The radioactive material labeled immunoassay (RIA) comprises reacting aseparated cell or crushing solution thereof, tissue or crushing solutionthereof, cell culture supernatant, serum, preural fluid, ascites fluid,ocular fluid or the like with the antibody of the present invention,further reacting the reactant with an anti-immunoglobulin antibody orbinding fragment labeled with radioisotope, and then measuring theradioactivity with a scintillation counter or the like.

The immunocyte staining and iminunotissue staining methods comprisereacting a separated cell, tissue or the like with the antibody of thepresent invention, reacting the reactant with an anti-immunoglobulinantibody or binding fragment labeled with a fluorescence substance suchas fluorescein isothiocyanate (FITC) or the like, or an enzyme such asperoxydase, biotin or the like, and then observing the cell, tissue orthe like with a microscope.

The sandwich ELISA is a method which comprises adsorbing, on a plate,one of two antibodies having a different epitope among the antibodies ofthe present invention; labeling another antibody with a fluorescencesubstance such as FITC or the like, or an enzyme such as peroxydase,biotin or the like; reacting a separated cell or crushing solutionthereof, tissue or crushing solution thereof, cell culture supernatant,serum, preural fluid, ascites fluid, ocular fluid, or the like with theantibody-adsorbing plate; and then reacting it with the labeled antibodyfor carrying out a reaction according to the labeled substance.

4. Diagnosis and Treatment of Th2-Mediated Immune Disease or Cancer

Since the humanized antibody of the present invention specifically bindsto CCR4 which is expressed on a cultured cell line and shows cytotoxicactivity such as CDC activity, ADCC activity and the like, it will beuseful in diagnosing and treating Th2-mediated diseases and the like.Also, since the proportion of amino acid sequences derived from humanantibody in the humanized antibody is higher than that in antibodies ofa non-human animal, it is expected that it shows strong cytotoxicactivity in the human body, it does not show immunogenicity, and itseffects continues for a long time.

In addition, the production of Th2 cytokines which are produced by cellssuch as IL-4, IL-5, IL-13 and the like, can be inhibited byadministering the antibody of the present invention to cells or tissuesof an experimental subject.

The Th2 cell used in the present invention is preferably activated Th2cell or memory Th2 cell. Specific examples include cells having CD45RA−and CD4+ properties.

The cytotoxic activities of the recombinant antibody of the presentinvention are generated, e.g., when the antibody of the presentinvention binds to a Th2 cell to thereby induce apoptosis in the cell.Also, the cell can be obstructed and depleted by inducing apoptosis.

Also, examples of the method for diagnosing Th2-mediated immune diseasesor cancers include a method in which a human CCR4 positive cell existingin cells or tissues of an experimental subject is immunologicallydetected as described above.

Furthermore, the antibody of the present invention can be used as adiagnostic agent for Th2-mediated immune diseases or cancers, ordiseases in which the morbid states advance due to abnormal increase ordecrease of Th2 cells.

Moreover, since the antibody of the present invention can reduce ordeplete CCR4-expressing cells by its cytotoxic activity, it can providea diagnostic method or therapeutic method for Th2-mediated immunediseases or cancers, which uses the antibody of the present invention,and therapeutic and preventive agents for Th2-mediated immune diseasesor cancers, which comprises the antibody of the present invention as anactive ingredient.

The Th2-mediated immune diseases include, irrespective of slight orserious, inflammatory diseases such as acute or chronic airwayhypersensitivity or bronchial asthma, atopic skin diseases includingatopic dermatitis, allergic rhinitis, pollinosis, and the like; diseasescaused by inflammation competent cells such as eosinophil, mast cell andthe like which can be propagated or activated by cytokine and chemokinereleased from Th2 cells, biologically functional molecules such as IgEand the like which are produced by cytokine and chemokine released fromTh2 cells, and the like; and immune diseases in which the morbid statesadvance due to abnormal changes in Th2 cells.

The antibody of the present invention can be administered alone, but itis generally preferred to provide it in the form of a pharmaceuticalformulation produced by mixing it with at least one pharmaceuticallyacceptable carrier in accordance with a method well known in thetechnical field of pharmaceutics.

It is preferred to select a route of administration which is the mosteffective in carrying out the intended treatment such as oraladministration or parenteral administration, e.g., intraoraladministration, tracheal administration, rectal administration,subcutaneous injection, intramuscular injection, intravenous injection,and the like. Intravenous injection is preferred in an antibody orpeptide formulation.

The dosage form includes sprays, capsules, tablets, granules, syrups,emulsions, suppositories, injections, ointments, tapes, and the like.

Examples of formulation suitable for oral administration includeemulsions, syrups, capsules, tablets, powders, granules, and the like.

Liquid preparations such as emulsions and syrups, can be produced usingadditives such as water; saccharides, e.g., sucrose, sorbitol, fructose,etc.; glycols, e.g., polyethylene glycol, propylene glycol, etc.; oils,e.g., sesame oil, olive oil, soybean oil, etc.; antiseptics, e.g.,p-hydroxybenzoate, etc.; flavors, e.g., strawberry flavor, peppermint,etc.; and the like.

Capsules, tablets, powders, granules and the like can be produced usingadditives such as fillers, e.g., lactose, glucose, sucrose, mannitol,etc.; disintegrating agents, e.g., starch, sodium alginate, etc.;lubricants, e.g., magnesium stearate, etc.; binders, e.g., polyvinylalcohol, hydroxypropylcellulose, gelatin, etc.; surfactants, e.g., fattyacid esters, etc.; plasticizers, e.g., glycerine, etc.; and the like.

Examples of formulations suitable for parenteral administration includeinjections, suppositories, sprays, and the like.

Injections can be prepared using a carrier such as a salt solution,glucose solution or a mixture thereof, or the like.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat, a carboxylic acid, or the like.

Also, sprays can be prepared from the antibody or peptide itself orusing a carrier or the like which does not stimulate oral and airwaymucous membranes of patients and can facilitate absorption of theantibody or peptide by dispersing it as minute particles.

Examples of the carrier include lactose, glycerine, and the like.Depending on the properties of the antibody or peptide and the carrierto be used, aerosols, dry powders and the like can be produced. Theadditives exemplified in the oral preparations can also be added to theparenteral preparations.

The dose and frequency of administration vary depending on intendedtherapeutic effect, administration method, treating period, age, bodyweight and the like, but the dose is generally from 10 μg/kg to 8 mg/kgper day per adult.

As discussed above, according to the present invention, a recombinantantibody and an antibody fragment thereof, which binds specifically tohuman CCR4 and contains novel CDRs for CCR4, are provided. The antibodyof the present invention is useful for the immunological detection of ahuman Th2 cell by immunocyte staining and for the diagnosis or treatmentof all Th2-mediated immune diseases including bronchial asthma andatopic skin diseases, diseases in which the morbid states advance due toabnormal balance of Th2 cells and cancers including blood cancers suchas leukemia.

Examples of the present invention are shown below, but the presentinvention is not limited thereto.

EXAMPLE 1

Production of Hybridoma Cell which Produces Mouse Anti-CCR4 MonoclonalAntibody:

Hybridoma cells which produce mouse anti-CCR4 monoclonal antibody KM2160(Int. Immunol., 11: 81 (1999)) were produced according to the followingprocedure.

(1) Preparation of Antigen

The amino acid sequence (SEQ ID NO:17) of human CCR4 (hereinafterreferred to as “hCCR4”) protein was analyzed by using Genetyx Mac, andCompound 1 (SEQ ID NO:1) was selected as a partial sequence consideredto be appropriate as the antigen among portions having highhydrophilicity, N-terminal and C-terminal. Also, a portion of the aminoacid sequence of a mouse CCR4 (hereinafter referred to as “mCCR4”)(BBRC, 218: 337 (1996)) protein corresponding to Compound 1 was selectedas Compound 2 (SEQ ID NO:2). SEQ ID NOs:1 and 2 correspond to positions2-29 from the N-terminal amino acids of human CCR4 and mouse CCR4,respectively.

Abbreviations of the amino acids and their protecting groups used in thepresent invention were used according to the recommendation by IUPAC-IUBJoint Commission on Biochemical Nomenclature (European Journal ofBiochemistry, 138: 9 (1984)).

Unless otherwise indicated, the following abbreviations represent thefollowing amino acids.

-   Ala: L-Alanine-   Asn: L-Asparagine-   Asp: L-Aspartic acid-   Asx: L-Aspartic acid or L-asparagine-   Cys: L-Cysteine-   Gln: L-Glutamine-   Glu: L-Glutamic acid-   Glx: L-Glutamic acid or L-glutamine-   Gly: Glycine-   Ile: L-Isoleucine-   Leu: L-Leucine-   Lys: L-Lysine-   Met: L-Methionine-   Phe: L-Phenylalanine-   Pro: L-Proline-   Ser: L-Serine-   Thr: L-Threonine-   Tyr: L-Tyrosine-   Val: L-Valine

The following abbreviations represent protecting groups of correspondingamino acids and side chain-protecting amino acids.

-   Fmoc: 9-Fluorenylmethyloxycarbonyl-   tBu: t-Butyl-   Trt: Trityl-   Boc: t-Butyloxycarbonyl-   Fmoc-Thr(tBu)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-threonine-   Fmoc-Ser(tBu)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-serine-   Fmoc-Tyr(tBu)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-tyrosine-   Fmoc-Lys(Boc)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-Nε-t-butyloxycarbonyl-L-lysine-   Fmoc-Asn(Trt)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-Nγ-trityl-L-asparagine-   Fmoc-Gln(Trt)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-Nδ-trityl-L-glutamine-   Fmoc-Asp(OtBu)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-L-aspartic acid β-t-butyl ester-   Fmoc-Glu(OtBu)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-L-glutamic acid γ-t-butyl ester-   Fmoc-Cys(Trt)-OH:    -   Nα-9-Fluorenylmethyloxycarbonyl-S-trityl-L-cysteine

The following abbreviations represent corresponding reaction solventsand reaction reagents.

-   PyBOP: Benzotriazol-1-yloxytripyrrolidinophosphonium    hexafluorophosphate-   HOBt: N-Hydroxybenzotriazole-   DMF: N,N-Dimethylformamide-   DCM: Dichloromethane-   TFA: Trifluoroacetic acid-   NMM: N-Methylmorpholine-   DTT: Dithiothreitol

(i) Synthesis of Compound 1 (SEQ ID NO:1)(H-Asn-Pro-Thr-Asp-Ile-Ala-Asp-Thr-Thr-Leu-Asp-Glu-Ser-Ile-Tyr-Ser-Asn-Tyr-Tyr-Leu-Tyr-Glu-Ser-Ile-Pro-Lys-Pro-Cys-OH)

Into a reaction vessel of an automatic synthesizer (manufactured byShimadzu), 30 mg of a carrier resin (chlorotrityl resin, manufactured byAnaSpec) to which 16.8 mmol of H-Cys(Trt) had been bound was placed, 1ml of DCM/DMF (1:1) was added thereto, followed by stirring for 10minutes, the solution was drained away, 1 ml of DMF was further added,followed by stirring for 1 minute, the solution was drained away, andthen the following procedure was carried out in accordance with thesynthesis program provided by Shimadzu.

(a) Fmoc-Pro-OH (168 μmol), PyBOP (168 μmol), HoBt.1H₂O (168 μmol) andNMM (252 μmol) were stirred in DMF (588.2 μl) for 5 minutes, theresulting solution was added to the resin, followed by stirring for 60minutes, and then the solution was drained away.

(b) The carrier resin was washed for 1 minute with 707 μl of DMF, andthis step was repeated 5 times. In this way, Fmoc-Pro-Cys(Trt) wassynthesized on the carrier.

Next, the following Fmoc group-deprotection steps were carried out.

(c) 707 μl of 30% piperidine-DMF solution was added, followed bystirring for 4 minutes, and then the solution was drained away, and thisprocedure was repeated again.

(d) The carrier resin was washed for 1 minute with 707 μl of DMF andthen the solution was drained away, and this step was repeated 5 times.

In this way, the carrier resin to which the Fmoc group-eliminatedH-Pro-Cys(Trt) had been bound was obtained.

Next, a condensation reaction was carried out in the step (a) usingFmoc-Lys(Boc)-OH, and then H-Lys(Boc)-Pro-Cys(Trt) was synthesized onthe carrier via the washing step of (b) and deprotection steps of (c)and (d). Next, the steps (a) to (d) were repeated by using Fmoc-Pro-OH,Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Tyr(tBu)-OH in this order in thestep (a). Next, the condensation reaction of step (a) was carried outusing Fmoc-Asn(Trt)-OH, the washing step of (b) was carried out, thecondensation reaction of step (a) using Fmoc-Asn(Trt)-OH and the washingstep of (b) were repeated and then the deprotection steps of (c) and (d)were carried out. Subsequently, the steps (a) to (d) were repeated byusing Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH,Fmoc-Glu(OtBu)-OH and Fmoc-Asp(OtBu)-OH in this order. Next, thecondensation reaction of step (a) was carried out using Fmoc-Leu-OH, thewashing step of (b) was carried out, the condensation reaction of step(a) using Fmoc-Leu-OH and the washing step of (b) were repeated and thenthe deprotection steps of (c) and (d) were carried out. Thereafter, thecondensation reaction steps of (a) and (b) were repeated twice by usingFmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ala-OH,Fmoc-Ile-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH andFmoc-Asn(Trt)-OH in that order in the step (a) and the deprotectionsteps of (c) and (d) were carried out, and then, after repetition of aseries of these steps, the mixture was washed with methanol and butylether in this order and dried under a reduced pressure for 12 hours toobtain the carrier resin to which a side chain-protected peptide hadbeen bound. A mixed solution (1 ml) of TFA (90%), thioanisole (5%) and1,2-ethanedithiol (5%) was added thereto, and incubated at roomtemperature for 2 hours to thereby remove the side chain protectinggroups and simultaneously cutting out the peptide from the resin. Afterremoving the resin by filtration, about 10 ml of ether was added to theresulting solution, and the thus formed precipitate was recovered bycentrifugation and decantation and dried under a reduced pressure toobtain 63.7 mg of crude peptide. The crude product was dissolved in 2 mlof DMF in the presence of 60 mg of DTT and then purified by HPLC using areverse phase column (CAPCELL PAK C18, 30 mm I.D.×25 mm, manufactured byShiseido). Elution was carried out according to a linear densitygradient method in which 90% acetonitrile aqueous solution containing0.1% TFA was added to 0.1% TFA aqueous solution and detecting at 220 nmwas carried out to obtain a fraction containing Compound 1. Afterfreeze-drying of this fraction, 2.5 mg of Compound 1 was obtained.

Mass spectrometry (FAB MS): m/z=3227.5 (M+H⁺)

-   Amino acid analysis: Asx 4.8 (5), Glx 2.7 (2), Ser 3.1 (3), Thr 2.0    (3), Ala 1.1 (1), Pro 3.1 (3), Tyr 3.8 (4), Leu 2.2 (2), Lys 1.2    (1), Ile 3.0 (3), Cys 1.2 (1)

(ii) Synthesis of compound 2 (SEQ ID NO: 2)(H-Asn-Ala-Thr-Glu-Val-Thr-Asp-Thr-Thr-Gln-Asp-Glu-Thr-Val-Tyr-Asn-Ser-Tyr-Tyr-Phe-Tyr-Glu-Ser- Met-Pro-Lys-Pro-Cys-OH)

Using 50 mg of a carrier resin (chlorotrityl resin, manufactured byAnaSpec) to which 28.0 μmol of H-Cys(Trt) had been bound as the startingmaterial, the steps (a) to (d) were repeated by using Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH andFmoc-Tyr(tBu)-OH in this order in the step (a) in the same manner as in(i). Next, the condensation reaction of step (a) was carried out usingFmoc-Ser(tBu)-OH, the washing step of (b) was carried out, thecondensation reaction of step (a) using Fmoc-Ser(tBu)-OH and the washingstep of (b) were repeated and then the deprotection steps of (c) and (d)were carried out. Next, the condensation reaction of step (a) wascarried out using Fmoc-Asn(Trt)-OH, the washing step of (b) was carriedout, the condensation reaction of step (a) using Fmoc-Asn(Trt)-OH andthe washing step of (b) were repeated and then the deprotection steps of(c) and (d) were carried out. Subsequently, the steps (a) to (d) wererepeated by using Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Thr(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Asp(OtBu)-OH in that order.Next, the condensation reaction of step (a) was carried out usingFmoc-Gln(Trt)-OH, the washing step of (b) was carried out, thecondensation reaction of step (a) using Fmoc-Gln(Trt)-OH and the washingstep of (b) were again repeated and then the deprotection steps of (c)and (d) were carried out. Thereafter, the condensation reaction steps of(a) and (b) were repeated twice by using Fmoc-Thr(tBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH and Fmoc-Asn(Trt)-OH inthis order in the step (a) and the deprotection steps of (c) and (d)were carried out, and then, after repetition of a series of these steps,the mixture was washed and dried to obtain the carrier resin to which aside chain-protected peptide was bound. A crude peptide (96.3 mg) wasobtained by eliminating the side chain protecting groups and cutting outthe peptide from the resin in the same manner as in (i), and furtherpurified by HPLC using a reverse phase column to obtain 5.8 mg ofCompound 2.

Mass spectrometry (TOFMS): m/z=3297.7 (M+H⁺)

-   Amino acid analysis: Asx 4.1 (4), Glx 4.3 (4), Ser 2.0 (2), Thr 4.6    (5), Ala 1.0 (1), Pro 2.2 (2), Tyr 3.9 (4), Val 1.9 (2), Met 1.0    (1), Phe 1.0 (1), Lys 1.1 (1), Cys 1.1 (1)    (2) Preparation of Immunogen

The hCCR4 partial peptide obtained in Example 1(1) was used as theimmunogen after preparing its conjugate with KLH (Calbiochem) by thefollowing method in order to increase its immunogenicity. That is, KLHwas dissolved in PBS to obtain 10 mg/ml, and 1/10 volume of 25 mg/ml MBS(Nakalai Tesque) was added dropwise thereto, followed by stirring for 30minutes. Free MBS was removed by a gel filtration column such asSephadex G-25 column which had been equilibrated in advance with PBS orthe like, and 2.5 mg of the resulting KLH-MB was mixed with 1 mg of thepeptide dissolved in 0.1 M sodium phosphate buffer (pH 7.0), followed bystirring at room temperature for 3 hours. After the reaction, themixture was dialyzed against PBS.

(3) Immunization of Animal and Production of Antibody Producing Cells

To 5-weeks-old female mice (Balb/c), 100 μg of the peptide-KLH conjugateprepared in Example 1(2) was administered together with 2 mg of aluminumgel and 1×10⁹ cells of pertussis vaccine (manufactured by Chiba SerumInstitute), and 2 weeks thereafter, 100 μg of the conjugate wasadministered once a week at a total of 4 times. A blood sample was takenfrom each animal from the venous plexus of the fundus of the eye, itsserum titer was examined by an enzyme immunoassay described below, andthe spleen was excised 3 days after the final immunization from a mousewhich showed a sufficient antibody titer. The spleen was excised from amouse on the 3rd day after the final administration and cut to pieces inMEM (manufactured by Nissui Pharmaceutical), and cells were unboundusing a pair of forceps and centrifuged (1,200 rpm, 5 minutes), thesupernatant was removed, followed by treatment with 3 ml of aTris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to eliminateerythrocytes. The remaining cells were further washed three times withMEM and used for cell fusion.

(4) Preparation of Mouse Myeloma Cell

An 8-azaguanine-resistant mouse myeloma cell line, P3X63Ag8U.1 (ATCCCRL-1597, hereinafter referred to as “P3-U1”), was cultured and used asthe parent line in cell fusion.

(5) Preparation of Hybridoma Cells

The spleen cells and myeloma cells obtained in Example 1(3) and (4) weremixed to a ratio of 10:1, followed by centrifuging (1,200 rpm, 5minutes) to remove the supernatant, 0.5 ml of a polyethylene glycolsolution (a solution containing 2 g of polyethylene glycol-1000, 2 ml ofMEM and 0.7 ml of DMSO) was added to the thus precipitated cells per 10⁸of spleen cells under conditions of 37° C., followed by thoroughlysuspending. Thereafter, 1 to 2 ml of MEM was added several times at 1 to2 minute intervals, and the final volume was adjusted to 50 ml with MEM.After removing the supernatant by centrifugation (900 rpm, 5 minutes),the precipitate was suspended in 100 ml of HAT medium, dispensed in 100μl/well into a 96 well microtiter plate (manufactured by SumitomoBakelite), followed by culturing in a 5% CO₂ incubator at 37° C. for 10to 14 days. Using wells in which propagation of the fused cell wasobserved, binding activity for the hCCR4 partial peptide (compound 1) inthe culture supernatant was measured by the ELISA described in 2(3) ofExample 2. Each well in which the activity was confirmed was cloned by atotal of 2 times of limiting dilution, once by changing the medium tothe HT medium and then changing the medium to the normal medium. In thisway, a hybridoma cell KM2160 which produces the mouse antibody KM2160was obtained. As shown in FIG. 1, KM2160 specifically reacted with thehCCR4 partial peptide (Compound 1).

EXAMPLE 2

Preparation of Anti-CCR4 Chimeric Antibody:

1. Isolation and Analysis of cDNA Encoding the V Region of Anti-CCR4Mouse Antibody:

(1) Preparation of mRNA from Hybridoma Cells which Produces Anti-CCR4Mouse Antibody

A mRNA was prepared from the hybridoma cell KM2160 described inExample 1. About 48 μg of mRNA was prepared from 8×10⁷ cells of thehybridoma cell KM2160 using a mRNA preparation kit, Fast Track mRNAIsolation Kit (manufactured by Invitrogen) according to themanufacture's instructions.

(2) Production of H Chain and L Chain cDNA Library of Anti-CCR4 MouseAntibody

cDNA having EcoRI-NotI adapters on both termini was synthesized from 5μg of the KM2160 mRNA obtained in 1(1) of Example 2 using cDNA Synthesiskit (manufactured by Amersham Pharmacia Biotech) according to themanufacture's instructions. The thus prepared cDNA was dissolved in 20μl of sterile water and fractionated by agarose gel electrophoresis, andabout 1.5 kb cDNA fragments corresponding to the H chain of IgG typeantibody and about 1.0 kb cDNA fragments corresponding to the L chain ofκ type were respectively recovered using QIAquick Gel Extraction Kit(manufactured by QIAGEN). Next, using λZAPII PredigestedEcoRI/CIAP-Treated Vector Kit (manufactured by Stratagene), each of 0.1μg of the about 1.5 kb cDNA fragments and 0.1 μg of the about 1.0 kbcDNA fragments was linked to 1 μg of the) λZAPII vector which had beendigested with a restriction enzyme EcoRI and terminus-dephosphorylatedwith Calf Intestine Alkaline Phosphatase according to the manufacture'sinstructions. A 2.5 μl portion of each reaction solution after theligation was packaged into λ phage using GigapackIII Gold PackagingExtract (manufactured by Stratagene) according to the manufacture'sinstructions, and then Escherichia coli line XL1-Blue (Biotechniques, 5:376 (1987)) was infected with an appropriate amount of the phage toobtain 9.3×10⁴ of phage clones as the H chain cDNA library of KM2160 and7.4×10⁴ of phage clones as the L chain cDNA library. Thereafter, eachphage was fixed on a nylon membrane filter Hybond-N+ (manufactured byAmersham Pharmacia Biotech) according to the manufacture's instructions.

(3) Cloning of H Chain and L Chain cDNAs of Anti-CCR4 Mouse Antibody

Using ECL Direct Nucleic Acid Labeling and Detection System(manufactured by Amersham Pharmacia Biotech), according to themanufacture's instructions, clones on the nylon membrane filters of theKM2160 H chain cDNA library and L chain cDNA library prepared in 1(2) ofExample 2 were detected using cDNA of the C region of a mouse antibody(H chain is a BamHI-EcoRI fragment of mouse Cγ1 cDNA (EMBO J., 3: 2047(1984)), L chain is a HpaI-EcoRI fragment of Cκ cDNA (Cell, 22: 197(1980)) as the probe, and phage clones strongly bound to the probe wereobtained as 10 clones for each of the H chain and the L chain. Next,each phage clone was converted into plasmid by the in vivo excisionmethod using λZAPII Cloning Kit according to the manufacture'sinstructions (manufactured by Stratagene). Using BigDye Terminator CycleSequencing FS Ready Reaction Kit (manufactured by PE Biosystems), thenucleotide sequence of cDNA contained in each plasmid obtained in thismanner was analyzed by a DNA sequencer ABI PRISM 377 of the samemanufacturer according to the manufacture's instructions. As a result, aplasmid pKM2160H4 containing a full length functional H chain cDNA and aplasmid pKM2160L6 containing a full length L chain cDNA, in which an ATGsequence considered to be the initiation codon is present in the 5′-endof cDNA, were obtained.

(4) Analysis of Amino Acid Sequence of V Region of Anti-CCR4 MouseAntibody

A full nucleotide sequence of the H chain V region contained in theplasmid pKM2160H4, a full amino acid sequence of the H chain V regiondeduced therefrom, a full nucleotide sequence of the L chain V regioncontained in the plasmid pKM2160L6 and a full amino acid sequence of theL chain V region deduced therefrom are shown in SEQ ID NOs:3, 15, 4 and16, respectively. Also, there are many nucleotide sequences whichcorrespond to the amino acid sequences represented by SEQ ID NOs:15 and16 other than those represented SEQ ID NOs:3 and 4, and all of them areincluded within the scope of the present invention. Based on thecomparison with sequence data of known mouse antibodies (Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)) and the comparison with the results of analysis of the H chainand L chain N-terminal amino acid sequences of the purified anti-CCR4mouse antibody KM2160 carried out using a protein sequencer (PPSQ-10,manufactured by Shimadzu), it was found that each cDNA thus isolated isa full length cDNA which encodes the anti-CCR4 mouse antibody KM2160containing secretory signal sequences which are amino acids of positions1-19 in the amino acid sequence shown in SEQ ID NO:15 in the H chain andamino acids of positions 1-19 in the amino acid sequence shown in SEQ IDNO:16 in the L chain.

Next, novelty of the amino acid sequences of the V regions of H chainand L chain of the anti-CCR4 mouse antibody KM2160 was examined. UsingGCG Package (version 9.1, manufactured by Genetics Computer Group) asthe sequence analyzing system, amino acid sequence data base of knownproteins were searched by BLAST method (Nucleic Acids Res., 25: 3389(1997)). As a result, completely coincided sequences were not found forboth of the H chain and L chain, so that it was confirmed that the Hchain V region and L chain V region of the anti-CCR4 mouse antibodyKM2160 are novel amino acid sequences.

Also, CDRs of the H chain V region and L chain V region of the anti-CCR4mouse antibody KM2160 were identified by comparing with amino acidsequences of known antibodies. Amino acid sequences of CDR1, CDR2 andCDR3 in the H chain V region of the anti-CCR4 mouse antibody KM2160 areshown in SEQ ID NOs:5, 6 and 7, respectively, and amino acid sequencesof CDR1, CDR2 and CDR3 in the L chain V region in SEQ ID NOs:8, 9 and10, respectively.

2. Stable Expression of Anti-CCR4 Chimeric Antibody Using Animal Cell

(1) Construction of Anti-CCR4 Chimeric Antibody Expression VectorpKANTEX2160

An anti-CCR4 chimeric antibody expression vector pKANTEX2160 wasconstructed as follows, using a humanized antibody expression vectorpKANTEX93 which expresses a human IgG1 and κ type antibody and theplasmids pKM2160H4 and pKM2160L6 obtained in 1(3) of Example 2.

A synthetic DNA having the nucleotide sequences shown in SEQ ID NOs:11and 12 was designed in order to obtain the H chain V region cDNA ofKM2160 by PCR, and another synthetic DNA having the nucleotide sequencesshown in SEQ ID NOs:13 and 14 for obtaining the L chain V region cDNA.Each synthetic DNA contains a restriction enzyme recognizing sequence inits 5′-end for its cloning into pKANTEX93, and synthesis of the DNA wasentrusted to Genset Inc. The plasmid pKM2160H4 (20 ng) obtained in 1(3)of Example 2 was added to a buffer containing 50 μl of PCR Buffer #1attached to KOD DNA Polymerase (manufactured by TOYOBO), 0.2 mM dNTPs, 1mM magnesium chloride and 0.5 μM of the synthetic DNA having thenucleotide sequences represented by SEQ ID NOs:11 and 12, and themixture was heated at 94° C. for 3 minutes. After 2.5 units of KOD DNAPolymerase (manufactured by TOYOBO) were added, the mixture wassubjected to 25 cycles of the reaction each cycle including heating at94° C. for 30 seconds, at 58° C. for 30 seconds and at 74° C. for 1minute, using a DNA thermal cycler GeneAmp PCR System 9600 (manufacturedby PERKIN ELMER). In the same manner, 20 ng of the plasmid pKM2160L6obtained in 1(3) of Example 2 was added to a buffer containing 50 μl ofPCR Buffer #1 attached to KOD DNA Polymerase (manufactured by TOYOBO),0.2 mM dNTPs, 1 mM magnesium chloride and 0.5 μM of the synthetic DNAhaving the nucleotide sequences represented by SEQ ID NOs:13 and 14, andPCR was carried out in the same manner as described above. The reactionsolution (10 μl) was subjected to agarose gel electrophoresis, and thenan H chain V region PCR product of about 0.46 kb and an L chain V regionPCR product of about 0.43 kb were each recovered using QIAquick GelExtraction Kit (manufactured by QIAGEN).

Next, 0.1 μg of DNA obtained by digesting a plasmid pBluescript SK(−)(manufactured by Stratagene) with a restriction enzyme SmaI(manufactured by Takara Shuzo) and about 0.1 μg of each of the PCRproducts obtained above were added to give 7.5 μl in final volume ofsterile water, and 7.5 μl of the solution I of TAKARA DNA Ligation KitVer. 2 (manufactured by Takara Shuzo) and 0.3 μl of a restriction enzymeSmaI were added thereto, and the mixture was allowed to react at 22° C.overnight. Using the resulting recombinant plasmid DNA solution, E. coliDH5α (manufactured by TOYOBO) was transformed. Each plasmid DNA wasprepared from the transformant clones and subjected to the reactionusing BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(manufactured by PE Biosystems) according to the manufacture'sinstructions, and the nucleotide sequence was analyzed by a DNAsequencer ABI PRISM 377 of the same manufacturer. Thus, the plasmidspKM2160VH41 and pKM2160VL61 shown in FIG. 2 having the desirednucleotide sequences were obtained.

Next, 3 μg of the humanized antibody expression vector pKANTEX93 and 3μg of the pKM2160VH41 obtained above were added to a buffer containing30 μl of 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT,10 units of a restriction enzyme ApaI (manufactured by Takara Shuzo)were added thereto, and the mixture was allowed to react at 37° C. for 1hour. The reaction solution was subjected to ethanol precipitation, andthe resulting precipitate was added to a buffer containing 10 μl of 50mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride,1 mM DTT, 100 μg/ml BSA and 0.01% Triton X-100, 10 units of arestriction enzyme NotI (manufactured by Takara Shuzo) were addedthereto, and the mixture was allowed to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis, andabout 12.75 kb and about 0.44 kb ApaI-NotI fragments of pKANTEX93 andpKM2160VH41, respectively, were recovered. The thus obtained twofragments were linked using TAKARA DNA Ligation Kit Ver. 2 according tothe manufacture's instructions, and E. coli DH5α (manufactured byTOYOBO) was transformed using the resulting recombinant plasmid DNAsolution. Each plasmid DNA was prepared from the transformant clones andconfirmed by a restriction enzyme treatment to thereby obtain a plasmidpKANTEX2160H shown in FIG. 3, in which about 0.44 kb of the desiredApaI-NotI fragment had been inserted.

Next, 3 μg of the pKANTEX2160H and 3 μg of the pKM2160VL61 obtainedabove were added to a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mMsodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml ofBSA, the solution was adjusted to give a total volume of 30 μl, 10 unitsof a restriction enzyme BsiWI (manufactured by New England Biolabs) wasadded thereto, and the mixture was allowed to react at 55° C. for 1hour. Then, a restriction enzyme EcoRI (manufactured by Takara Shuzo)was added thereto, and the mixture was allowed to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 13.20 kb and about 0.41 kb EcoRI-BsiWIfragments of pKANTEX2160H and pKM2160VL61, respectively, were recovered.The thus obtained two fragments were linked using TAKARA DNA LigationKit Ver. 2 according to the manufacture's instructions, and E. coli DH5α(manufactured by TOYOBO) was transformed using the resulting recombinantplasmid DNA solution. Each plasmid DNA was prepared from thetransformant clones and confirmed by a restriction enzyme treatment tothereby obtain a plasmid pKANTEX2160 shown in FIG. 4, in which about0.41 kb of the desired EcoRI-BsiWI fragment had been inserted. When theplasmid was subjected to the reaction using BigDye Terminator CycleSequencing FS Ready Reaction Kit (manufactured by PE Biosystems)according to the manufacture's instructions, and the nucleotide sequencewas analyzed by a DNA sequencer ABI PRISM 377 of the same manufacturer,it was confirmed that the desired plasmid into which cDNA encoding theKM2160 H chain and L chain V regions had been cloned was obtained.

(2) Stable Expression of Anti-CCR4 Chimeric Antibody Using Animal Cell

The anti-CCR4 chimeric antibody was expressed in animal cells asdescribed below using the anti-CCR4 chimeric antibody expression vectorpKANTEX2160 obtained in 2(1) of Example 2.

The plasmid pKANTEX2160 was converted into a linear form by digestingwith a restriction enzyme AatII (manufactured by TOYOBO) and 10 μgthereof was introduced into 4×10⁶ cells of rat myeloma cell line YB2/0(ATCC CRL1662) by electroporation (Cytotechnology, 3: 133 (1990)), andthe cells were suspended in 40 ml of H-SFM (manufactured by GIBCO-BRL)medium (supplemented with 5% FCS) and dispensed in 200 μl/well into a 96well microtiter plate (manufactured by Sumitomo Bakelite). Twenty-fourhours after incubation at 37° C. in a 5% CO₂ incubator, G418 was addedto give a concentration of 1 mg/ml, followed by culturing for 1 to 2weeks. A culture supernatant was recovered from a well in which a colonyof G418-resistant transformant appeared and became confluent, andantigen-binding activity of the anti-CCR4 chimeric antibody in thesupernatant was measured by ELISA shown in 2(3) of Example 2 (aperoxidase-labeled goat anti-human IgG(γ) antibody was used as thesecondary antibody).

In order to increase the expressed amount of the antibody using a dhfrgene amplification system, the transformant in a well where expressionof the anti-CCR4 chimeric antibody was found in the culture supernatantwas suspended to give a density of 1 to 2×10⁵ cells/ml in H—SFM mediumcontaining 1 mg/ml G418 and 50 nM methotrexate (hereinafter referred toas “MTX”: manufactured by Sigma) which is the inhibitor of a dhfr geneproduct dihydrofolate reductase (hereinafter referred to as “DHFR”), andthe suspension was dispensed in 1 ml into wells of a 24 well plate(manufactured by Greiner). The mixture was cultured at 37° C. for 1 to 2weeks in a 5% CO, incubator, so that a transformant showing resistanceto 50 nM MTX was induced. When the transformant became confluent in awell, antigen-binding activity of the anti-CCR4 chimeric antibody in theculture supernatant was measured by ELISA shown in 2(3) of Example 2.Regarding the transformants in wells where expression of the anti-CCR4chimeric antibody was found in culture supernatants, the MTXconcentration was increased to 100 nM and then to 200 nM in the samemanner to finally obtain a transformant which can grow in H—SFM mediumcontaining 1 mg/ml of G418 and 200 nM of MTX and can also highly expressthe anti-CCR4 chimeric antibody. The thus obtained transformant wassubjected to single cell isolation (cloning) by two times of limiteddilution assay, and a transformant clone having the highest anti-CCR4chimeric antibody expression was named KM2760. The expressed amount ofthe anti-CCR4 chimeric antibody by KM2760 was about 5 μg/10⁶ cells/24hours. In addition, the antibody H chain C region of KM2760 belongs tohuman IgG1 subclass. KM2760 has been internationally deposited as FERMBP-7054 on Feb. 24, 2000, in National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, the Ministry ofInternational Trade and Industry (present name: International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology) (Higashi 1-1-3, Tsukuba-shi, Ibaraki Prefecture, Japan).

(3) Measurement of Binding Activity of Antibody to CCR4 Partial Peptide(ELISA)

The hCCR4 partial peptide (Compound 1) obtained in Example 1(1) wasconjugated with thyroglobulin (hereinafter referred to as “THY”) andused as the antigen for the assay. The production method was asdescribed in Example 1(2), except that4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC, manufactured by Sigma) was used instead of MBS as thecrosslinking agent. The conjugate prepared in the above manner wasdispensed at 10 μg/ml and 50 μl/well into a 96 well plate for EIA(manufactured by Greiner) and adhered thereto by incubating it at 4° C.overnight. After washing with PBS, 1% BSA-containing PBS (hereinafterreferred to as “1% BSA-PBS”) was added at 100 μl/well, and the mixturewas allowed to react at room temperature for 1 hour to block theremaining active groups. After removing 1% BSA-PBS, diluted solutions ofa transformant culture supernatant, a purified mouse antibody or apurified human chimeric antibody was dispensed at 50 μl/well, and themixture was allowed to react at room temperature for 1 hour. After thereaction, each well was washed with 0.05% Tween 20-containing PBS(hereinafter referred to as “Tween-PBS”), a peroxidase-labeled rabbitanti-mouse Ig antibody solution (manufactured by DAKO) diluted 400 timeswith 1% BSA-PBS and a peroxidase-labeled goat anti-human IgG(γ) antibodysolution (manufactured by American Qualex) diluted 3,000 times with 1%BSA-PBS were dispensed into the mouse antibody-added wells and the humanchimeric antibody-added wells, respectively, as the secondary antibodysolution at 50 μl/well, and the mixture was allowed to react at roomtemperature for 1 hour. After the reaction, each well was washed withTween-PBS, ABTS solution (a solution prepared by dissolving 0.55 g of2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium in 1liter of 0.1 M citrate buffer (pH 4.2), and adding 1 μl/ml of hydrogenperoxide just before use) was dispensed at 50 μl/well for colordeveloping, and the absorbance at 415 nm (hereinafter referred to as“OD₄₁₅”) was measured.

(4) Purification of Anti-CCR4 Chimeric Antibody from Culture Supernatant

The transformant cell clone KM2760 which expresses the anti-CCR4chimeric antibody obtained in 2(2) of Example 2 was suspended in H—SFM(manufactured by GIBCO-BRL) containing 200 nM MTX and 5% Daigo's GF21(manufactured by Wako Pure Chemical Industries) to give a density of 1to 2×10⁵ cells/ml, and dispensed at 100 ml into 175 cm² flasks(manufactured by Greiner). The cells were cultured at 37° C. for 5 to 7days in a 5% CO₂ incubator, and the culture supernatant was recoveredwhen they became confluent. The anti-CCR4 chimeric antibody KM2760 waspurified from about 200 ml of the culture supernatant using Prosep-A(manufactured by Bioprocessing) column according to the manufacture'sinstructions to thereby obtain about 1.9 mg of the purified protein.About 3 μg of the resulting anti-CCR4 chimeric antibody KM2760 wasapplied to electrophoresis according to the known method (Nature, 227:680 (1970)) to examine its molecular weight and purification degree. Theresults are shown in FIG. 5. As shown in FIG. 5, the purified anti-CCR4chimeric antibody KM2760 was about 150 kilodaltons (hereinafter referredto as “Kd”) under non-reducing conditions, and two bands of about 50 Kdand about 25 Kd were observed under reducing conditions. The sizes ofthe proteins almost coincided with the molecular weights deduced fromthe cDNA nucleotide sequences of H chain and L chain of KM2760 (H chain:49,226, L chain: 24,168) and also coincided with reports disclosing thatIgG type antibody has a molecular weight of about 150 Kd undernon-reducing conditions and is degraded into an H chain having amolecular weight of about 50 Kd and an L chain having a molecular weightof about 25 Kd under reducing conditions due to cutting of theintramolecular disulfide bond (hereinafter referred to as “S—S bond”)(Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter14 (1988), Monoclonal Antibodies: Principles and Practice, AcademicPress Limited (1996)), so that it was confirmed that the anti-CCR4chimeric antibody KM2760 was expressed as the antibody molecule having acorrect structure. Also, the N-terminal amino acid sequences of the Hchain and L chain of the purified anti-CCR4 chimeric antibody KM2760were analyzed using a protein sequencer (PPSQ-L0, manufactured byShimadzu), it was confirmed that they coincide with N-terminal aminoacid sequences of the H chain and L chain of the anti-CCR4 mouseantibody KM2160.

3. Establishment of hCCR4-High-Expressing Cell

(1) Construction of Expression Vector CAG-pcDNA3 for Animal Cell

An expression vector was constructed as descried below by producing anexpression vector (CAG-pcDNA3) in which the promoter region of anexpression vector for animal cell, pcDNA3 (manufactured by INVITROGEN),was changed from cytomegalovirus (CMV) promoter to CAG (AG (modifiedchicken βactin) promoter with CMV-IE enhancer), and inserting the CCR4gene into the vector.

pcDNA3 (5 μg) was allowed to react with a restriction enzyme NruI(manufactured by Takara Shuzo) at 37° C. for 1 hour, and then DNAfragments were recovered by ethanol precipitation. Next, they wereallowed to react with a restriction enzyme HindIII (manufactured byTakara Shuzo) at 37° C. for 1 hour and then fractionated by agarose gelelectrophoresis to recover a DNA fragment of about 5.8 kb containing noCMV promoter region. Plasmid CAG-pBluescript IIKS(+) (3 μg) having CAGpromoter (Nuc. Acid. Res., 23: 3816 (1995)) region was allowed to reactwith a restriction enzyme SalI (manufactured by Takara Shuzo) at 37° C.for 1 hour and then DNA fragments were recovered by ethanolprecipitation. They were blunt-ended with DNA Blunting Kit (manufacturedby Takara Shuzo), further allowed to react with HindIII at 37° C. for 1hour, and then fractionated by agarose gel electrophoresis to recover aDNA fragment of about 1.8 kb containing the CAG promoter region. Thethus recovered respective DNA fragments were ligated using DNA LigationKit (manufactured by Takara Shuzo), and E. coli DH5α was transformedusing the resulting recombinant plasmid DNA to obtain plasmidCAG-pcDNA3.

(2) Construction of hCCR4 Expression Vector

An hCCR4 expression vector was constructed as described below by usingthe CAG-pcDNA3 obtained in 3(1) of Example 2 and hCCR4DNA-insertedpcDNA3 (CCR4/pcDNA3). Both of the CAG-pcDNA3 and CCR4/pcDNA3 wereallowed to react with HindIII at 37° C. for 1 hour and DNA fragmentswere recovered by ethanol precipitation. Next, they were allowed toreact with BglII (manufactured by Takara Shuzo) at 37° C. for 1 hour andthen fractionated by agarose gel electrophoresis to recover a DNAfragment of about 2.0 kb containing the CAG promoter region and a DNAfragment of about 5.5 kb containing the hCCR4 gene region. Thereafter,plasmid CAG-CCR4/pcDNA3 was obtained using both of the DNA fragments inthe same manner as in 3(1) of Example 2.

(3) Expression of hCCR4 in Animal Cell

The plasmid was introduced into animal cells by electroporation in thesame manner as described in 2(2) of Example 2. EL-4 cells (ATCC TIB-39)were suspended in PBS(−) (manufactured by GIBCO-BRL) to give a densityof 1×10⁷ cells/500 μl, 10 μg of the CAG-CCR4/pcDNA3 obtained in 3(2) ofExample 2 was added thereto, and the mixture was incubated in ice for 10minutes and then put into a cuvette for exclusive use (manufactured byBio-Rad) to carry out gene introduction at 260 V and 500 μFD. After themixture was further incubated in ice for 10 minutes, the cells weresuspended in 200 ml of 10% FCS-RPMI medium and dispensed at 200 μl/wellinto a 96 well plate for cell culturing. Twenty-four hours afterculturing, 100 μl of the culture supernatant was removed from each well,and 10% FCS-RPMI medium containing 1 mg/ml of G418 was dispensed at 100μl/well to give a final concentration of 0.5 mg/ml. Two weeksthereafter, single clones of between 10 and 100 were selected andcultured again.

(4) Selection of hCCR4-High-Expressing Cell

They were selected by an immunofluorescent method using KM2160 preparedin Example 1(5). Into a 96 well U shape plate, 2×10⁵ cells of each ofselected several tens of the gene-introduced clones was dispensed.KM2160 labeled with biotin by a known method (Enzyme Antibody Method,published by Gakusai Kikaku) was diluted to 5 μg/ml with a buffer forFACS (1% BSA-PBS, 0.02% EDTA, 0.05% NaN₃, pH 7.4), human IgG(manufactured by Welfide) was diluted to 3.75 mg/ml to preventnonspecific staining, each of the thus diluted antibody solution wasdispensed at 200 μl/well, and the mixture was allowed to react in icefor 30 minutes. As a negative control, biotinylated anti-IL-SR antibody(WO 97/10354) was used at the same concentration. After washing twicewith 200 μl/well of the buffer, streptoavidin-PE (manufactured by BectonDickinson Japan) was dispensed at 20 μl/well. Thirty minutes after thereaction in ice in the dark, the cells were washed three times with 200μl/well and finally suspended to 500 μl and the fluorescence intensitywas measured by a flow cytometer to select one cell line having thehighest fluorescence intensity.

EXAMPLE 3

Analysis of Function of Anti-CCR4 Chimeric Antibody:

1. Evaluation of Activity of Anti-CCR4 Chimeric Antibody

(1) Reactivity of Anti-CCR4 Chimeric Antibody for Human and Mouse CCR4(ELISA)

Reactivity of the purified anti-CCR4 chimeric antibody KM2760 to humanCCR4 and mouse CCR4 was measured by ELISA shown in 2(3) of Example 2.The hCCR4 partial peptide (Compound 1) and mCCR4 partial peptide(Compound 2) obtained in Example 1(2) were conjugated with THY and usedas the antigen. The preparation was carried out in the same manner as inExample 1(2), except that SMCC (manufactured by Sigma) was used insteadof MBS as the crosslinking agent. FIG. 6 shows a result of theexamination of reactivity in which the CCR4 peptide conjugate to beadhered was fixed to each well of a ELISA plate to 10 μg/ml and 50μl/well and the concentration of the anti-CCR4 chimeric antibody KM2760to be added was changed. As shown in FIG. 6, the anti-CCR4 chimericantibody KM2760 had almost binding activity to the hCCR4 partial peptideand mCCR4 partial peptide at a similar level. Based on this result, itwas found that the anti-CCR4 chimeric antibody KM2760 recognizes theepitope existing in a region of positions 2-29 from the N-terminal aminoacid of human CCR4 and mouse CCR4.

(2) Reactivity of Anti-CCR4 Chimeric Antibody with hCCR4-High-ExpressingCells (Immunofluorescent Method)

Reactivity of the purified anti-CCR4 chimeric antibody KM2760 with thehCCR4-high-expressing cell (hereinafter referred to as “CCR4/EL-4”)produced in 3(4) of Example 2 was measured in the same manner as in 3(4)of Example 2. As shown in FIG. 7, the anti-CCR4 chimeric antibody KM2760showed strong reactivity with CCR4/EL-4 cells.

2. In Vitro Cytotoxic Activity of Anti-CCR4 Chimeric Antibody (ADCCActivity)

In order to evaluate in vitro cytotoxic activity of the purified CCR4chimeric antibody obtained in 2(4) of Example 2, its ADCC activity wasmeasured as described below.

(1) Preparation of Target Cell Suspension

The hCCR4-high-expressing cell CCR4/EL-4 obtained in 3(4) of Example 2was cultured in 10% FCS-RPMI 1640 medium containing 0.5 mg/ml G418 togive a density of 1×10⁶ cells/0.5 ml, 1.85 MBq equivalent of radioactivesodium chromate (Na₂ ⁵¹CrO₄) (manufactured by Daiichi Pure Chemicals)was added thereto and the mixture was allowed to react at 37° C. for 1.5hours to thereby isotope-label the cells. After the reaction, the cellswere washed three times by their suspension in RPMI 1640 medium andcentrifugation, re-suspended in the medium and then incubated in ice at4° C. for 30 minutes to thereby spontaneously release the radioactivesubstance. After centrifugation, 5 ml of 10% FCS-RPMI 1640 medium wasadded thereto to give a density of 2×10⁵ cells/ml, and the mixture wasused as the target cell suspension.

(2) Preparation of Effector Cell Suspension

Healthy human peripheral blood (60 ml) was collected using a syringecontaining 200 units (200 μl) of a heparin sodium injection(manufactured by Takeda Pharmaceutical). The entire amount was filled upto 120 ml by diluting it two times with the same volume of physiologicalsaline (manufactured by Otsuka Pharmaceutical). Lymphoprep (manufacturedby NYCOMED) was dispensed at 5 ml into 12 tubes of 15 ml capacitycentrifugation tubes (manufactured by Sumitomo Bakelite), the dilutedperipheral blood was over-layered thereon at 10 ml, and the mixture wascentrifuged at room temperature and 800×g for 20 minutes. PBMC fractionsbetween the blood plasma layer and the Lymphoprep layer were collectedfrom all centrifugation tubes, suspended in 1% FCS-containing RPMI 1640medium (manufactured by GIBCO) (hereinafter referred to as “1%FCS-RPMI”), washed twice by centrifugation at 400×g and 4° C. for 5minutes and then re-suspended to give a density of 5×10⁶ cells/ml to beused as the effector cells.

(3) Measurement of ADCC Activity

The target cell suspension prepared in 2(1) of Example 3 was dispensedat 50 μl (1×10⁴ cells/well) into wells of a 96 well U bottom plate(manufactured by Falcon). Next, the effector cell suspension prepared in2(2) of Example 3 was dispensed at 100 μl (5×10⁵ cells/well, the ratioof effector cells to target cells becomes 50:1). Subsequently, eachanti-CCR4 chimeric antibody was added to give a final concentration of0.01 to 10 μg/ml and the mixture was allowed to react at 37° C. for 4hours. After the reaction, the plate was centrifuged and the amount of⁵¹Cr in 100 μl of the supernatant in each well was measured by aγ-counter. The amount of the spontaneously dissociated ⁵¹Cr wascalculated in the same manner as the above using the medium aloneinstead of the effector cell suspension and antibody solution andmeasuring the amount of ⁵¹Cr in the supernatant. The amount of the totaldissociated ⁵¹Cr was calculated in the same manner as the above byadding the medium alone instead of the antibody solution, and 1 Nhydrochloric acid instead of the effector cell suspension, and measuringthe amount of ⁵¹Cr in the supernatant. The ADCC activity was calculatedby the following equation:

${{Cytotoxic}\mspace{14mu}{activity}\mspace{14mu}(\%)} = {\frac{\begin{matrix}{\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{in}\mspace{14mu}{sample}\mspace{14mu}{supernatant}} \right) -} \\\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{spontaneously}\mspace{14mu}{released}\mspace{14mu}{\,^{51}{Cr}}} \right)\end{matrix}}{\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{total}\mspace{14mu}{\,^{51}{Cr}}} \right) - \left( {{spontaneously}\mspace{14mu}{released}\mspace{14mu}{\,^{51}{Cr}}} \right)} \times 100}$

The results are shown in FIG. 8. As shown in FIG. 8, the anti-CCR4chimeric antibody showed strong cytotoxic activityantibody-concentration-dependently within a range of 0.01 to 10 μg/ml.When ADCC activity of a cell line into which no gene was incorporated,EL-4 cells, was measured in the same manner as a negative control, theactivity was not found, so that it was confirmed that the cytotoxicactivity is CCR4-specific. The above results show that the anti-CCR4chimeric antibody KM2760 can reduce or deplete CCR4-expressed Th2 cellsby efficiently activating human effector cells and therefore is usefulfor diagnosis or treatment of Th2-mediated immune diseases in human suchas bronchial asthma, atopic skin inflammation, and the like.

EXAMPLE 4

Effect of Anti-CCR4 Chimeric Antibody on Human Peripheral Blood:

(1) Measurement of ADCC Activity on Human PBMC

PBMC was isolated in the same manner as in 2(2) of Example 3, andsuspended to give a final density of 1×10⁷ cells/ml. The mixture wasdispensed at 100 μl/well into the U plate used in 2(3) of Example 3 togive a density of 1×10⁶ cells/well. Each of KM2760 and a negativecontrol anti-IL-5 receptor antibody (WO 97/10354) was diluted to give aconcentration of 20 μg/ml and dispensed at 100 μl/well into thecell-dispensed wells. Twenty-four hours after culturing at 37° C. in astream of 5% CO₂, the cells were recovered. The cytotoxicity wasdetected using Annexin V-EGFP Apoptosis Detection Kit (manufactured byMBL), and annexin V-positive cells were considered to be dead cells. Thecells were centrifuged at 4° C. for 3 minutes at 400×g and suspended inthe binding buffer attached to the kit to give a density of 5×10⁵cells/200 μl. The suspension was mixed with 1 μl of the Annexin V-EGFPreagent, followed by pipetting several times, and then the mixture wasallowed to react for 5 minutes under shading. After the reaction, themixture was centrifuged at 4° C. for 3 minutes at 400×g, and thesupernatant was removed. The pellet was loosened by lightly stirring ona vortex mixer, 100 μl of 2.5 mM CaCl₂-containing methanol which hadbeen cooled on ice was added thereto, and the mixture was incubated at4° C. for 10 minutes. The mixture was centrifuged for 30 seconds at8,000×g, and the supernatant was removed. The precipitate was suspendedin 200 μl of the binding buffer and washed twice by centrifugation. Tothe remaining pellet after removing the supernatant, 10 μl ofPC5-labeled anti-CD4 antibody (manufactured by Coulter), 20 μl ofPE-labeled anti-CD45RA antibody (manufactured by Coulter) and 20 μl ofFACS buffer containing 2.5 mM CaCl₂ were added, and the mixture wasallowed to react at 4° C. for 30 minutes. Thereafter, the mixture waswashed three times by centrifugation using the FACS buffer containing2.5 mM CaCl₂ and analyzed by a flow cytometer (manufactured by Coulter).First, the group of cells was fractionated into 4 fractions(CD4+CD45RA+, CD4+CD45RA−, CD4−CD45RA+ and CD4−CD45RA−) based on thedifference in staining properties between CD4 and CD45RA. Next, theannexin V-positive ratio in each fraction was measured and representedas cytotoxicity %. The results are shown in FIG. 9. The cytotoxicity ofPBMC was observed only when co-cultured with KM2760, and the toxicitywas detected specifically in the CD4+CD45RA− fraction to which CCR4−positive cells belong.

(2) Effect of Inhibiting Production of Cytokine from Human PBMC

In the same manner as described in 2(3) of Example 3, ADCC activity wasinduced by co-culturing PBMC and KM2760 (final concentration: 10 μg/ml)for 24 hours. After culturing, 100 μl of the supernatant was removed and100 of a medium containing 1 μg/ml PMA (phorbol myristate acetate) and200 ng/ml A23187 (calcimycin: manufactured by RBI) was added insteadthereof, so that final concentrations of PMA and A23187 became 0.5 μg/mland 100 ng/ml, respectively, and then the cytokine production wasinduced by stimulating the cells (Condition (i)). As another stimulationconditions, the cytokine production was induced using 50 ng/ml at afinal concentration of an anti-CD3 antibody OKT-3 (ATCC CRL-8001)instead of A23187 (Condition (ii)). Each stimulating agent wasintroduced, and then, twenty-four hours after culturing, the culturesupernatant was recovered to measure IL-4, IL-5, IL-13 and interferon(IFN)-γ using a cytokine measuring kit (manufactured by BioSource). Asshown in FIG. 10, production of a Th2 cytokine IL-4, IL-5 or IL-13 wasinhibited in the KM2760-added group, but production of a Th1 cytokineIFN-γ was not influenced.

EXAMPLE 5

Reactivity of Anti-CCR4 Chimeric Antibody with Human T Cell LeukemiaCell Line:

(1) Binding Ability to Membrane Surface (Immunofluorescent Method)

Reactivity of the anti-CCR4 chimeric antibody KM2760 to the following 8human T cell leukemia cell lines: HPB-ALL (Cancer Research, 54: 1511(1994); acute T cell leukemia), HSB-2 (ATCC CCL-120.1; leukemia (Tcell)), MOLT-4 (JCRB9031; lymphoma (T cell)), TALL-1 (JCRB 0086;leukemia (T cell)), Jurkat (ATCC TIB-152; acute T cell leukemia),CCRF-CEM (ATCC CCL-119; acute T cell leukemia), PEER (JCB 0830; leukemia(T cell)) and Hut78 (ATCC TIB-161; cutaneous T cell lymphoma), wasmeasured in the same manner as in 3(4) of Example 2. In this case, theconcentration of the biotinylated KM2760 was changed to 10 μg/ml. Asshown in FIG. 11, the anti-CCR4 chimeric antibody KM2760 showed strongreactivity with 5 lines (HPB-ALL, Jurkat, CCRF-CEM, PEER and Hut78)among the 8 lines tested.

(2) ADCC Activity

ADCC activity for the five cell lines whose reactivity with theanti-CCR4 chimeric antibody KM2760 had been confirmed in 1 of Example 5was measured in the same manner as in 2 of Example 3. As shown in FIG.12, the anti-CCR4 chimeric antibody KM2760 injured all of the testedcells concentration-dependently.

EXAMPLE 5

Evaluation of In Vivo Activity of Anti-CCR4 Chimeric Antibody KM2760(Inhibition of Th2 Cytokine Production):

In order to evaluate in vivo activity of the purified anti-CCR4 chimericantibody KM2760 obtained in 2(4) of Example 2, the antibody wasadministered to Macaca fascicularis by single intravenous injection andblood samples were periodically collected for about 1 month, cytokineproduction was induced in the peripheral leukocytes by stimulation withPMA (Phorbol 12-Myristate 13-Acetate, manufactured by Sigma) andIONOMYCIN (manufactured by Sigma), and Th2 cytokines, IL-4 and IL-13,and Th1 cytokine, IFN-γ, were measured. The methods and results aredescribed below.

The dose (protein content) of the purified anti-CCR4 chimeric antibodyKM2760 was 0.1 mg/kg, 1.0 mg/kg or 10 mg/kg, and the antibody wasadministered to 2 animals of four-year-old male Macaca fascicularis bysingle intravenous injection in each of the dose groups. Blood sampleswere collected from the femoral vein before the administration and onthe 1st, 2nd, 3rd and 4th weeks after the administration. Heparin(Heparin Sodium Injection, 1,000 units/ml, manufactured by ShimizuPharmaceutical) was used as the anticoagulant and added to 1 ml of bloodto give a concentration of 25 units/ml. Using a flat bottom 24 wellplate, peripheral blood of each individual was dispensed at 500 μl/wellinto 2 wells. A medium containing the stimulating agent (PMA finalconcentration 50 ng/ml, IONOMYCIN final concentration 1 μg/ml) was addedto one well, and the medium containing no stimulating agent to anotherwell, each at 500 μl/well, and the mixture was lightly stirred andcultured at 37° C., 5% CO, and 95% air for 24 hours. Also, the mediumused was prepared by adding 0.5 ml of a penicillin-streptomycin solution(manufactured by GIBCO BRL) and 5.6 ml of immobilized fetal calf serum(manufactured by PAA Laboratories) to 100 ml of RPMI 1640 (GIBCO BRL).After completion of culturing, the culture broth containing blood cellswas recovered from each well and centrifuged (6,700×g, 5 minutes, 4° C.)to obtain a supernatant. IL-4, IL-13 and IFN-γ contained in theresulting culture supernatant were determined using respective cytokinemeasuring kits (IL-4: OptEIA Human IL-4 Kit, manufactured by PharMingen;IL-13: Cyto screen Human IL-13 Immunoassay Kit, manufactured byBioSource International; IFN-γ: Cyto screen Human IFN-γ Immunoassay Kit,manufactured by BioSource International). The produced amount ofcytokine by each individual is a value calculated by subtracting theamount obtained by not adding the stimulating agent from the amountobtained by adding the stimulating agent (0.1 mg/kg group, individualNos. L-1 and L-2; 1.0 mg/kg group, individual Nos. M-1 and M-2; 10 mg/kggroup, individual Nos. H-1 and H-2). The results are shown in FIGS. 13to 15. In these drawings, the value of each of IL-4, IL-13 and IFN-γ ineach individual before the administration was used as 100%, and theproduced amount after the administration was shown by percentage. Th2cytokines, IL-4 (FIG. 13) and IL-13 (FIG. 14), significantly decreasedon the 1st week after the administration in all administered groups, andthe inhibition continued even on the 4th week after the administration.On the other hand, influence upon Th1 cytokine, IFN-γ (FIG. 15), wasextremely small.

Based on these results, it was found that production of Th2 cytokinefrom peripheral blood mononuclear cells is inhibited at least for 4weeks when the anti-CCR4 chimeric antibody KM2760 is administered toMacaca fascicularis. It was also shown that the anti-CCR4 chimericantibody KM2760 reduced or depleted CCR4-expressing Th2 cells inperipheral blood in the body of Macaca fascicularis.

EXAMPLE 6

In Vivo Antitumor Activity of Anti-CCR4 Chimeric Antibody:

(1) Antitumor effect of anti-CCR4 chimeric antibody KM2760 on syngenicintraperitoneal graft model

Antitumor effect of the anti-CCR4 chimeric antibody KM2760 on a mousesyngenic tumor model in which the hCCR4-high-expressing mouse-derivedCCR4/EL-4 cells obtained in 2(4) of Example 2 was grafted into theabdominal cavity of mouse was measured. Eight-weeks-old male C57BL/6mice (CLEA Japan) were used. The CCR4/EL-4 cells were suspended inPBS(−) (manufactured by Gibco BRL) to give a density of 1×10⁵ cells/mland grafted into the abdominal cavity of each of 10 C57BL/6 mice at adose of 200 μl/animal. Four hours, three days, six days and ten daysafter the transplantation, 200 μl of KM2760 diluted to 2 mg/ml with acitrate buffer (an aqueous solution of 10 mmol/l citric acid and 150mmol/l sodium chloride, adjusted to pH 6) was administered into theabdominal cavity of each of 5 animals among them. The remaining 5animals were used as a negative control group to which the chimericantibody was not administered. The number of days from the day oftransplantation until mice of each group died due to proliferation oftumor cells accompanied by ascites is shown in Table 1. The averagenumber of survival days was 16.4 day in the negative control group,whereas the average number of survival days in the KM2760-administeredgroup was 26.2 day. Since 59.8% of survival period prolongation wasfound by the KM2760 administration, KM2760 has a life prolongationeffect on the syngenic intraperitoneal graft model of CCR4 expressingleukemia cells.

TABLE 1 Average value Survival ratio Days survived (day) (%) Negativecontrol 14/14/16/18/20 16.4 group KM2760-administered 21/22/23/29/3626.2 59.8 group(2) Antitumor Effect of Anti-CCR4 Chimeric Antibody KM2760 on SyngenicSubcutaneous Graft Model

Antitumor effect of the anti-CCR4 chimeric antibody KM2760 on a mousesyngenic tumor model in which the hCCR4-high-expressing mouse-derivedCCR4/EL-4 cells obtained in 2(4) of Example 2 was subcutaneously graftedinto a mouse was measured. Eight-weeks-old male C57BL/6 mice (CLEAJapan) were used. The CCR4/EL-4 cells were suspended in PBS(−)(manufactured by Gibco BRL) to give a density of 1×10⁶ cells/ml andgrafted under the ventral side skin of each of 18 C57BL/6 mice at a doseof 50 μl/animal. Regarding 5 animals among them, 100 μl of KM2760diluted to 2 mg/ml with a citrate buffer (an aqueous solution of 10mmol/l citric acid and 150 mmol/l sodium chloride, adjusted to pH 6) wasadministered in the tail vein after 4 hours of the transplantation oncea day for continuous 5 days. In this case, the dose per administrationis 200 μg/animal. Regarding another 6 animals, 4 hours, 7 days and 14days after the transplantation, 200 μl of KM2760 diluted to 2 mg/ml withthe citrate buffer was administered in the tail vein. In this case, thedose per administration is 400 μg/animal. The remaining 7 animals wereused as a negative control group to which the chimeric antibody was notadministered. Six days after the transplantation, tumor diameter wasperiodically measured using slide calipers, and the antitumor effect wasjudged by the ratio of the average value of tumor volume in eachadministered group to the average value of tumor volume in eachnon-administered group and by the surviving days after commencement ofthe administration. The tumor volume was calculated by the followingequation:Tumor volume=(breadth)²×length×0.5

The average value of tumor volumes in each group is shown in Table 2 andFIG. 16, and results of the ratio of the average value of tumor volumesin each administered group to the average value of tumor volumes in thenon-administered group are shown in Table 3 and FIG. 17. The ratio ofthe average value of tumor volumes in each KM2760-administered group tothe average value of tumor volumes in the non-administered group on the18th day after the transplantation was 0.356 in the group in which 200μg was administered continuously for 5 days and 0.257 in the group inwhich 400 μg was administered three times, so that KM2760 showed agrowth inhibition effect on the syngenic subcutaneous graft model ofCCR4-positive leukemia cells by each of the administration schedules.

TABLE 2 Days after transplantation Group constitution 0 6 10 12 15 18Negative control 50 ± 0 46 ± 28 294 ± 114 778 ± 263 1825 ± 708  3581 ±1279 group KM2760 200 μg × 50 ± 0 16 ± 20 115 ± 60  299 ± 194 601 ± 4291274 ± 886  5 days continuous administration group KM2760 400 μg × 50 ±0 0 ± 0 73 ± 49 198 ± 158 431 ± 467  920 ± 1163 three timesadministration group Unit: mm³ ± standard deviation

TABLE 3 Days after transplantation Group constitution 0 6 10 12 15 18Negative control group 1.000 1.000 1.000 1.000 1.000 1.000 KM2760 200 μg× 5 days 1.000 0.348 0.393 0.384 0.330 0.356 continuous administrationgroup KM2760 400 μg × three times 1.000 0.000 0.250 0.254 0.236 0.257administration group(3) Antitumor Effect of Anti-CCR4 Chimeric Antibody KM2760 on XenograftModel

Antitumor effect of the anti-CCR4 chimeric antibody KM2760 on a mousesubcutaneous tumor xenograft model in which the hCCR4-expressing human Tcell leukemia line CCRF-CEM cell (ATCC CCL-119) was subcutaneouslygrafted into nude mouse was measured. Eight-weeks-old male Balb/c nudemice (CLEA Japan) were used. The CCRF-CEM cells were suspended in RPMI1640 medium (manufactured by Gibco BRL) to give a density of 1×10⁸cells/ml and grafted under the ventral side skin of each of 20 Balb/cnude mice at a dose of 200 μl/animal. Thereafter, they were divided into4 groups of 5 animals per group, and 200 μl of KM2760 diluted to 2mg/ml, 0.5 mg/ml or 0.2 mg/ml with the citrate buffer was administeredto the three groups in the tail vein after 4 hours, 3 days and 6 days ofthe transplantation. In this case, the administered doses of KM2760 inrespective groups are 400 μg/animal/day, 100 μg/animal/day and 40μg/animal/day. The remaining one group was used as a negative controlgroup by administering 200 μl of human immunoglobulin G (hereinafterreferred to as “hIgG”, manufactured by Welfide) diluted to 2 mg/ml withthe citrate buffer in the tail vein (400 μg/animal/day). Four days afterthe transplantation, the tumor diameter was periodically measured usingslide calipers, and the antitumor effect was judged by the tumor volumein each group. The tumor volume was calculated by the followingequation:Tumor volume=(breadth)²×length×0.5

Changes with time in the average values of tumor volume in each groupare shown in Table 4 and FIG. 18. Complete inhibition of tumor growthwas observed in all of the KM2760-administered groups, so that KM2760showed a growth inhibition effect on the subcutaneous tumor xenograftmodel of CCR4-positive leukemia cells.

TABLE 4 Days after transplantation Group constitution 0 6 10 12 15 18Negative control 0 ± 0 44 ± 63 204 ± 159 233 ± 157 364 ± 86  448 ± 142group KM2760 40 μg 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 administeredgroup KM2760 100 μg 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 administeredgroup KM2760 400 μg 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 administeredgroup Unit: mm³ ± standard deviation

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one of skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof. All references cited hereinare incorporated in their entirety.

What is claimed is:
 1. A method for producing a recombinant antibody oran antibody fragment thereof that specifically binds to the epitoperecognized by mouse antibody KM2160 produced by FERM BP-10090 and thathas antibody-dependent cell-mediated cytotoxic activity against a cellwhich expresses CCR4 comprising: culturing a cell capable of producingthe recombinant antibody or an antibody fragment thereof thatspecifically binds to the epitope recognized by mouse antibody KM2160.2. The method of claim 1, wherein said recombinant antibody or antibodyfragment thereof is a humanized antibody or a human antibody.
 3. Themethod of claim 2, wherein the humanized antibody or fragment thereof isa human chimeric antibody or a human CDR-grafted antibody.
 4. The methodof claim 2, wherein the antibody is a IgG antibody.
 5. The method ofclaim 2, wherein the humanized antibody comprises: a CDR1, CDR2 and CDR3of an antibody H chain V region having the amino acid sequences setforth in SEQ ID NOS:5, 6 and 7, respectively; and a CDR1, CDR2 and CDR3of an antibody L chain V region having the amino acid sequences setforth in SEQ ID, NOS:8, 9 and 10, respectively.
 6. The method of claim3, wherein the human chimeric antibody comprises: an antibody H chain Vregion and antibody L chain V region of a monoclonal antibody whichspecifically reacts with CCR4; and an H chain C region and L chain Cregion of a human antibody.
 7. The method of claim 3, wherein the humanchimeric antibody comprises: a CDR1, CDR2 and CDR3 of an H chain Vregion having the amino acid sequences set forth in SEQ ID NOS:5, 6 and7, respectively; and CDR1, CDR2 and CDR3 of an L chain V region havingthe amino acid sequences set forth in SEQ ID NOS:8, 9 and 10,respectively.
 8. The method of claim 3, wherein the human chimericantibody comprises: an H chain V region having amino acids of positions20-138 in the amino acid sequence set forth in SEQ ID NO:15; and an Lchain V region having amino acids of positions 20-132 in the amino acidsequence set forth in SEQ ID NO:
 16. 9. The method of claim 3, whereinthe human chimeric antibody is an antibody KM2760 produced by atransformant KM2760 (FERM BP-7054), and wherein its antibody H chain Cregion belongs to human IgG1 subclass.
 10. The method of claim 3,wherein the human CDR-grafted antibody comprises: CDRs of an antibody Hchain V region and an antibody L chain V region of a monoclonal antibodywhich specifically reacts with CCR4; and C regions of an H chain and anL chain and a V region framework region of a human antibody.
 11. Themethod of claim 3, wherein the human CDR-grafted antibody comprises:CDR1, CDR2 and CDR3 of an H chain V region having the amino acidsequences set forth in SEQ ID NOS:5, 6 and 7, respectively; and CDR1,CDR2 and CDR3 of an L chain V region having the amino acid sequences setforth in SEQ ID NOS:8, 9 and 10, respectively.
 12. The method of claim2, wherein the human antibody comprises an antibody H chain V region andan antibody L chain V region.
 13. The method of claim 12, wherein CDRsof the H chain V region and L chain V region of the human antibodycomprise amino acid sequences which are the same as amino acid sequencesof CDRs of an H chain V region and an L chain V region, respectively, ofa monoclonal antibody which specifically reacts with CCR4.
 14. Themethod of claim 12, wherein the human antibody comprises: CDR1, CDR2 andCDR3 of an H chain V region having the amino acid sequences set forth inSEQ ID NOS:5, 6 and 7, respectively, and CDR1, CDR2 and CDR3 of an Lchain V region having the amino acid sequences set forth in SEQ IDNOS:8, 9 and 10, respectively.
 15. The method of claim 12, wherein the Hchain V region and L chain V region of the human antibody comprise aminoacid sequences which are the same as amino acid sequences of an H chainV region and an L chain V region, respectively, of a monoclonal antibodywhich specifically reacts with CCR4.
 16. The method of claim 15, whereinthe human antibody comprises: an H chain V region having amino acids ofpositions 20-138 in the amino acid sequence set forth in SEQ ID NO: 15;and an L chain V region having amino acids of positions 20-132 in theamino acid sequence set forth in SEQ ID NO:
 16. 17. The method of claim12, wherein the human antibody is an antibody obtained from a humanantibody phage library or a transgenic animal which produces a humanantibody.
 18. The method of claim 1, wherein said recombinant antibodyor said fragment is a Fab, Fab′, F(ab′)2, a single chain antibody, or adisulfide stabilized V region fragment.
 19. The method of claim 18,wherein the recombinant antibody fragment comprises the antibody H chainV region and an antibody L chain V region of an antibody.
 20. The methodof claim 19, wherein the CDRs of the H chain V region and L chain Vregion of the recombinant antibody fragment comprise amino acidsequences which are the same as amino acid sequences of CDRs of an Hchain V region and an L chain V region, respectively, of a monoclonalantibody which specifically reacts with CCR4.
 21. The method of claim20, wherein the CDRs of the H chain V region and L chain V region of therecombinant antibody fragment comprise: CDR1, CDR2 and CDR3 of the Hchain V region having the amino acid sequences set forth in SEQ IDNOS:5, 6 and 7, respectively; and CDR1, CDR2 and CDR3 of the L chain Vregion having the amino acid sequences set forth in SEQ ID NOS:8, 9 and10, respectively.
 22. The method of claim 19, wherein the H chain Vregion and L chain V region of the recombinant antibody fragmentcomprise amino acid sequences which are the same as amino acid sequencesof an H chain V region and an L chain V region, respectively, of amonoclonal antibody which specifically reacts with CCR4.
 23. The methodof claim 22, wherein an H chain V region having amino acids of positions20-138 in the amino acid sequence set forth in SEQ ID NO:15; and an Lchain V region having amino acids of positions of 20-132 in the aminoacid sequence set forth in SEQ ID NO:
 16. 24. A method of producing aconjugated recombinant antibody or an antibody fragment thereof, whichspecifically reacts with an extracellular domain of human CCR4,comprising: culturing a cell capable of producing a recombinant antibodyor an antibody fragment thereof that specifically binds to the epitoperecognized by mouse antibody KM2160; and chemically or geneticallyconjugating said antibody or said fragment with a radioisotope, aprotein or an agent.
 25. The method of claim 1, further comprisingcombining the recombinant antibody or the antibody fragment thereof witha pharmaceutically acceptable carrier thereby producing a medicament.26. The method of claim 3, wherein said human CDR-grafted antibodycomprises: CDR1, CDR2 and CDR3 of an H chain V region, and CDR1, CDR2and CDR3 of an L chain V region having the amino acid sequences setforth in SEQ ID NOS:5-10, respectively.
 27. The method of claim 3,wherein the human antibody comprises: CDR1, CDR2 and CDR3 of an H chainV region, and CDR1, CDR2 and CDR3 of an L chain V region having theamino acid sequences set forth in SEQ ID NOS:5-10, respectively.
 28. Themethod of claim 1, wherein said cell is a cell selected from the groupconsisting of a SP2/0-Ag14 cell, mouse P3X63-Ag8.653 cell, CHO cell, CHOcell in which a dihydrofolate reductase gene is defective, YB23HL.P2.G11.16Ag.20 cell, NSO cell, COS cell, human myeloma cell and aCHO/DG44 cell.